MY-ANH NGUYEN - University of Ottawa€¦ · MY-ANH NGUYEN . Thesis submitted to the Faculty of...

Characterization of PCSK9-mediated LDLR Degradation in Hepatic and Fibroblast Cells

MY-ANH NGUYEN

Thesis submitted to the Faculty of Graduate and Postdoctoral Studies

In partial fulfillment of the requirements

For the degree of

MASTER of SCIENCE

Department of Biochemistry, Microbiology and Immunology

University of Ottawa

Ottawa, Ontario, Canada

© My-Anh Nguyen, Ottawa, Canada, June 2013

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Abstract

The discovery that proprotein convertase subtilisin/kexin type 9 (PCSK9) mediates

degradation of low-density lipoprotein receptors (LDLR) indicates a critical role in LDL

metabolism. PCSK9 is a secreted protein that binds to the epidermal growth factor-like

(EGF)-A domain of LDLR and directs the receptor for degradation in lysosomes by an

unknown mechanism. A gain-of-function mutation, D374Y, increases binding to LDLR

EGF-A >10-fold and is associated with a severe form of hypercholesterolemia in humans.

Similar to previous studies, data obtained in my project has established that PCSK9 was

capable of promoting robust LDLR degradation in liver-derived cell lines; however, minimal

effects on LDLR levels were detected in several lines of fibroblast cells despite normal

LDLR-dependent cellular uptake of PCSK9. Importantly, a PCSK9 degradation assay

showed that 125I-labeled wild-type PCSK9 was internalized and degraded equally in both

hepatic and fibroblast cells, indicating dissociation of wild-type PCSK9 from recycling

LDLRs in fibroblasts. Moreover, PCSK9 recycling assays confirmed that no recycling of

wild-type PCSK9 to the cell surface could be detected in fibroblast cells. In contrast, more

than 60% of internalized PCSK9-D374Y recycled to the cell surface in these cells, and thus

had reduced ability to direct the LDLR for lysosomal degradation despite persistent binding.

Co-localization studies indicated that PCSK9-D374Y trafficked to both lysosomes and

recycling compartments in fibroblast cells, whereas wild-type PCSK9 exclusively trafficked

to lysosomes. We conclude that two factors diminish PCSK9 activity in fibroblast cells: i) an

increased dissociation from the LDLR in early endosomal compartments, and ii) a decreased

ability of bound PCSK9 to direct the LDLR to lysosomes for degradation. Finally, an LDLR

variant that binds to PCSK9 in a Ca2+-independent manner could partially restore wild-type

PCSK9 activity, but not PCSK9-D374Y activity, in fibroblast cells.

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Acknowledgments “Đi một ngày đàng, học một sàng khôn” is a famous expression in my mother-tongue

language, Vietnamese. It means the more you go, the more you learn. At least, it’s absolutely right in my case. Coming to Canada and becoming a graduate student at University of Ottawa Heart Institute have really been the biggest turning point in my life. I have grown a lot, both on a personal and professional level. Two years are not long time but also not short time, especially if you have to live far away from your loved ones, if you have to leave all you’ve known and restart your life from the beginning. Luckily, I have many special people around who give me constant strength, motivation, and encouragement to keep going the way I have chosen. First of all, I know that I always can count on my family. I don’t dedicate this achievement for anyone of my family because for me, each of them is meaningful on their own. When everything gets me down, just some simple things such as my grandmother’s voice, unlimited chatting with my sisters, funny stories of my brother, or sweet hugs from my aunt are enough to make my day. I can be sure that sometimes nobody in my family can understand what I am doing or what I am talking about, but they are always there for me whenever I need. My life is never complete without them.

Importantly, I want to express my special thanks to Dr. Thomas Lagace. Tom is a perfect supervisor who not only knows how to firmly push you forward but also knows how to gently help you realize your valuable abilities and develop them. I can honestly say that I have learnt a lot from him. From the bottom of my heart, I am really grateful to him for his endless patience and guidance. He was always willing to listen to me, even many of my crazy opinions or silly questions, to clear my mind when I was confused, and to bring me back the right way when I got lost. He never gave up on me no matter how many times I made him disappointed.

Besides, I am very grateful to a number of PIs especially for their assistance and guidance that I needed most during this program. I would like to thank Dr. Xiaohui Zha and Pasan Fernando, who were my TAC member over the years, for giving me critical discussions as well as valuable advices about my project. I also want to thank Dr. Ross Milne who always supported me in a special way. His constant encouragement usually made me stronger to overcome many difficulties throughout, more hopeful to complete my project, and more determined to pursue my further goals in science.

There will be a mistake if I don’t say thanks to many people who I am lucky enough to work with. First of all are my wonderful labmates: Geoff Leblond and Tanja Kosenko who trained me at the very beginning and always devoted their time as well as effort to assist me whenever I needed; Mia Golder who was always there to teach me anything I did not know, to explain anything I did not understand, and to show me anything I needed to improve. I cannot remember how many times they helped me get out of my problems, even minor or major. All of them are my best teachers. Also, thanks to Maroun Khalil who showed me how to go through countless headaches related to confocal microscope (for sure, he is one of whole-hearted and patient mentors who I have ever got), to Suzanne Crowe who helped me with flow cytometry experiments, to every people at the fourth floor of the Heart Institute who was always available for help with chemical, equipment or anything and everything. Thanks all of you for making my graduate student experiences at the Heart Institute more memorable.

To my dearest friends who always stayed beside me and supported me during this period of my life, I love all of you so much and thank for always being my true friends no matter how freaky I am sometimes.

Finally, thanks to you, my special one, although you always say that there’s no need to say thanks between us and I never can express enough thanks to you.

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Contents

Abstract ................................................................................................................................... ii

Acknowledgments ................................................................................................................. iii

List of Abbreviations Used .................................................................................................. vii

List of Figures ........................................................................................................................ ix

1 Introduction ........................................................................................................................ 1

1.1 Regulation of cholesterol metabolism .................................................................... 2

1.1.1 Cholesterol, the central lipid of mammalian cells .................................. 2

1.1.2 SREBPs: main regulators of cholesterol homeostasis............................ 3

1.2 LDLR and LDL uptake ........................................................................................... 6

1.2.1 LDLR ..................................................................................................... 6

1.2.2 LDLR-mediated LDL uptake pathway .................................................. 9

1.3 PCSK9: a new player in cholesterol metabolism ................................................. 11

1.3.1 Structure and processing of PCSK9 ..................................................... 11

1.3.2 The crystal structure of PCSK9 ............................................................ 13

1.3.3 Regulation of PCSK9 ........................................................................... 15

1.4 PCSK9-mediated LDLR degradation ................................................................... 16

1.4.1 Crystal structure of LDLR/PCSK9 complex ........................................ 17

1.4.2 Molecular characterization of PCSK9-mediated LDLR degradation ......

............................................................................................................. 21

1.4.3 Sites of action ....................................................................................... 25

1.5 Research Objectives .............................................................................................. 27

2 Materials and Methods .................................................................................................... 29

2.1 Materials ............................................................................................................... 29

2.1.1 Chemicals and reagents ........................................................................ 29

2.1.2 Antibodies ............................................................................................ 29

2.2 Protein assay ......................................................................................................... 30

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2.3 Purification of human wild-type PCSK9 and PCSK9(D374Y)-FLAG fusion

proteins ................................................................................................................. 30

2.4 Tissue culture medium ......................................................................................... 31

2.5 Protein labeling .................................................................................................... 31

2.5.1 AlexaFuor 488- labeled proteins ....................................................... 31

2.5.2 Biotin-labeled proteins ......................................................................... 32

2.6 PCSK9 cellular uptake assay ................................................................................ 32

2.7 Biotinylation and immunoblot analysis ................................................................ 33

2.8 LDLR degradation assay on 917 and HuH7 cells ................................................ 34

2.9 125I-radiolabeled PCSK9 degradation assay ......................................................... 34

2.10 Recycling assay .................................................................................................. 36

2.10.1 Transferrin ............................................................................................ 36

2.10.2 PCSK9 .................................................................................................. 36

2.11 Live cell imaging for co-localization studies ..................................................... 37

2.11.1 PCSK9 and LysoTracker ...................................................................... 37

2.11.2 PCSK9 and transferrin ......................................................................... 37

2.11.3 Transferrin and Rab4 ............................................................................ 38

2.12 Mutagenesis ....................................................................................................... 38

2.13 Transient transfection......................................................................................... 39

2.14 Statistical analysis .............................................................................................. 39

3 Results ............................................................................................................................... 40

3.1 Exogenous PCSK9 significantly decreased LDLR levels in HepG2 hepatic cells,

not in SV589 fibroblast cells ................................................................................ 40

3.1.1 PCSK9 at physiological concentrations ............................................... 40

3.1.2 PCSK9 at higher concentrations .......................................................... 42

3.2 Long incubation time did not interfere with the resistance of SV589 fibroblast

cells to PCSK9-mediated LDLR degradation ...................................................... 44

3.3 PCSK9 was ineffective to degrade LDLRs in another fibroblast cell line, 917

foreskin fibroblasts ............................................................................................... 46

3.4 PCSK9 endocytosis was LDLR-dependent in both hepatic and fibroblast cells . 49

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3.5 Both wild-type PCSK9 and the mutant PCSK9-D374Y trafficked to lysosomes in

hepatic cells .......................................................................................................... 52

3.6 Wild-type PCSK9, not the mutant PCSK9-D374Y, was completely degraded in

fibroblast cells ...................................................................................................... 56

3.7 More internalized PCSK9-D374Y proteins recycled to the cell surface.............. 58

3.7.1 Recycling assay .................................................................................... 58

3.7.2 Co-localization studies ......................................................................... 61

3.8 LDLR-EGF66 variant that binds PCSK9 in a calcium-independent manner could

restore wild-type PCSK9 activity in fibroblast cells ............................................ 66

4 Discussion .......................................................................................................................... 69

4.1 PCSK9 degrades LDLRs in a cell-dependent manner ......................................... 69

4.2 PCSK9 association/uptake is LDLR-dependent in both of hepatic and fibroblast

cells ....................................................................................................................... 71

4.3 PCSK9 in hepatic cells ......................................................................................... 72

4.4 PCSK9 in fibroblast cells ..................................................................................... 73

4.5 A possible role of endosomal calcium concentrations in PCSK9-mediated LDLR

degradation ........................................................................................................... 77

4.6 Future directions ................................................................................................... 80

4.7 Conclusion ............................................................................................................ 82

5 References ......................................................................................................................... 84

6 Curriculum Vitae ............................................................................................................. 96

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List of Abbreviations

ABCA1 ATP-binding cassette transporter

ADH Autosomal dominant hypercholesterolemia

AE Adverse effect

Apo Apolipoprotein ApoER2 Apolipoprotein E receptor 2

AP-2 Adaptor protein 2

ARH Autosomal recessive hypercholesterolemia

bHLH-Zip Basic helix-loop-helix leucine zipper

BAPTA 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid

BCA Bicinchoninic acid

BSA Bovine albumin serum

CHD Coronary heart disease

COPII Coat protein complex II

EGF Epidermal growth factor

ER Endoplasmic reticulum

ESCRT Endosomal sorting complex required for trafficking

FBS Fetal bovine serum

FH Familial hypercholesterolemia

HDL High-density lipoprotein

HMGCR 3-hydroxyl-3-methylglutaryl coenzyme reductase

HRS Hepatocyte growth factor-regulated Tyr-kinase substrate

INSIG-1 Insulin-induced gene 1

LA LDLR type-A

LDL Low-density lipoprotein

LDL-C Low-density lipoprotein cholesterol

LDLR Low-density lipoprotein receptor

LRP8 Low-density lipoprotein receptor-related protein 8

LXR Liver X receptor

NARC-1 Neural apoptosis regulated convertase 1

NCLPDS Newborn calf lipoprotein-deficient serum

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PCSK9 Proprotein convertase subtilisin/kexin type 9

Rab4 RAS-related GTP-binding protein 4

RAP Receptor-associated protein

S1P Site-1 protease

S2P Site-2 protease

SCAP SREBP cleavage-activating protein

SIRT1 Sirtuin 1

SRE Sterol response element

SREBP Sterol regulatory element binding protein

TCA Trichloroacetic acid

TCEP Tris(2-carboxyethyl)phosphine hydrochloride

TfR Transferrin receptor

TSG101 Tumor susceptibility gene 101

VLDL Very low-density lipoprotein

VLDLR Very low-density lipoprotein receptor

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List of Figures

Figure 1.1 The SREBP pathway ......................................................................................... 5

Figure 1.2 Cellular itinerary of the LDLR .......................................................................... 8

Figure 1.3 Structure of PCSK9 ......................................................................................... 12

Figure 1.4 Model of LDLR degradative pathway mediated by secreted PCSK9 in hepatic

cells .................................................................................................................. 18

Figure 1.5 The LDLR-PCSK9 complex at acidic pH ....................................................... 19

Figure 3.1 Addition of PCSK9 at physiologically relevant concentrations causes robust

LDLR degradation in HepG2 human hepatoma cells, but not SV589 human

fibroblast cells ................................................................................................. 41

Figure 3.2 Changes of endogenous LDLR levels in SV589 human fibroblast cells when

PCSK9 was added at higher concentrations .................................................... 43

Figure 3.3 Changes of endogenous LDLR levels in HepG2 human hepatoma cells when

PCSK9 was added at higher concentrations .................................................... 45

Figure 3.4 Increased incubation time with PCSK9 had no effects on the resistance of

SV589 human fibroblast cells to PCSK9-mediated LDLR degradation ......... 47

Figure 3.5 PCSK9-mediated LDLR degradation assay in HuH7 human hepatoma cells

and 917 human fibroblast cells ........................................................................ 48

Figure 3.6 PCSK9 uptake in the presence and absence of LDLRs in HepG2 human

hepatoma cells and SV589 human fibroblast cells .......................................... 51

Figure 3.7 PCSK9 uptake in cells expressing either wild-type LDLR or a PCSK9

binding-defective mutation .............................................................................. 53

Figure 3.8 125I-labeled PCSK9 degradation assay ............................................................ 55

Figure 3.9 Co-localization of PCSK9 and the lysosome marker (LysoTracker) in HepG2

human hepatoma cells ..................................................................................... 57

Figure 3.10 Recycling assay of wild-type PCSK9 and PCSK9-D374Y in SV589 human

fibroblast cells ................................................................................................. 59

Figure 3.11 Co-localization of wild-type PCSK9 and the lysosome marker (LysoTracker)

in SV589 human fibroblast cells ..................................................................... 63

Figure 3.12 Co-localization of PCSK9-D374Y and the lysosome marker (LysoTracker) in

SV589 human fibroblast cells.......................................................................... 64

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Figure 3.13 Co-localization of the mutant PCSK9-D374Y and transferrin in recycling

compartments of SV589 human fibroblast cells ............................................. 65

Figure 3.14 Wild-type LDLR and LDLR-EGF66 degradation mediated by PCSK9 in

SV589 human fibroblast cells.......................................................................... 68

Figure 4.1 Model of wild-type PCSK9 in fibroblast cells ................................................ 75

Figure 4.2 Model of PCSK9-D374Y in fibroblast cells.................................................... 76

Figure 4.3 Binding of PCSK9-R194 to LDLR-D310 in the calcium-binding site of the

LDLR EGF-A domain ..................................................................................... 78

1

1. Introduction

Coronary heart disease (CHD), which leads to one of every five deaths each year,

remains a persistent health burden in westernized countries (1). The link between CHD and

lipids has been firmly established; in which elevated levels of plasma cholesterol is

considered as the leading risk factor for CHD. Lowering cholesterol levels, particularly

circulating low-density lipoprotein cholesterol (LDL-C) has been the focus of the prevention

of CHD and its sequels for almost 35 years (2). While 3-hydroxyl-3-methylglutaryl

coenzyme reductase (HMGCR) inhibitors (statins) are the most effective drugs to treat

cardiovascular diseases in many groups, their potential adverse effects (AE), such as muscle

AEs (myositis as well as rhabdomyolysis) and other nonmuscle AEs (pancreatic and hepatic

dysfunction), should be considered (3).

The discovery that proprotein convertase subtilisin/kexin type 9 (PCSK9) functions as

a strong negative regulator of hepatic low-density lipoprotein receptor (LDLR) levels

suggested a critical role in low-density lipoprotein (LDL) metabolism (4-7). Importantly,

gain-of-function mutations in PCSK9 lead to hypercholesterolemia and premature

atherosclerosis, while loss-of-function mutations result in lowered plasma LDL-C levels and

significant protection from CHD without apparent AEs on other aspects of human

physiology (8). Thus, PCSK9 is a validated therapeutic target for cholesterol lowering.

Blocking antibodies raised against PCSK9 that prevent the LDLR interaction are actively

being developed by pharmaceutical companies, and some are now entering Phase III clinical

trials to lower plasma cholesterol levels in patients (9, 10). However, less invasive and cost

effective alternatives are still sought, therefore it is important to determine the mechanisms

of PCSK9-mediated LDLR degradation in the endolysosomal system.

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1.1 Regulation of cholesterol metabolism

1.1.1 Cholesterol, the central lipid of mammalian cells

Cholesterol has been extensively studied since the French chemist, M. E. Chevreul,

discovered it in 1815. For over 100 years, cholesterol’s structure, biosynthetic pathways as

well as feedback mechanisms regulating cholesterol metabolism have been revealed and

characterized (11).

Mammalian cells acquire cholesterol endogenously as well as exogenously. Whereas

extrahepatic tissues obtain most of their cholesterol from de novo biosynthesis, in

hepatocytes, most cellular cholesterol is derived from the circulation in the form of

apolipoprotein (Apo) B-containing lipoproteins such as LDL (12). Cholesterol is synthesized

in the endoplasmic reticulum (ER) and cytoplasm from the two-carbon building block,

acetyl-CoA, through the mevalonate pathway (13). The cholesterol biosynthetic pathway has

been considered as a complex pathway related to more than 40 cytosolic and membrane-

bound enzymes. At least 14 enzymes in this biosynthetic pathway are subjected to feedback

regulation by cellular cholesterol levels, of which the rate-limiting enzyme is HMGCR that

catalyzes the conversion of HMG-CoA to mevalonate and is the common target for

cholesterol lowering drugs such as statins as mentioned above (14, 15). After the first

committed step in cholesterol synthesis that converts squalene to the first sterol (lanosterol),

cholesterol is synthesized by a series of oxidation, reduction and demethylation reactions

(16, 17). There are evidences for two alternative pathways in the last steps of cholesterol

synthesis that differ in the point at which Δ24 double bond is reduced. Both 7-

dehydrocholestrol and desmosterol have been demonstrated to be the immediate precursors

of cholesterol (18, 19).

In addition to de novo synthesis, cells can obtain cholesterol via the LDLR-mediated

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uptake of plasma lipoproteins (20). Enterocytes of the small intestine play a key role in

absorbing and packaging dietary cholesterol, along with triglycerides, into chylomicrons.

When chylomicrons reach the circulation via the lymph, lipoprotein lipase hydrolyzes some

of the triglycerides, and the chylomicron remnants will be taken up by hepatocytes.

Hepatocytes also secrete lipids in the form of very low-density lipoprotein (VLDL) particles

that are processed in the circulation into LDL, the main lipoprotein that delivers cholesterol

to the peripheral cells (21). LDL along with other ApoE/ApoB-containing lipoproteins is

subsequently taken up through LDLR-mediated endocytosis (22).

Among many lipids in mammalian cells, cholesterol has an undeniable importance.

Based on its biophysical properties, cholesterol serves as a vital constituent of mammalian

cell membranes. Particularly, cholesterol condenses and rigidifies bilayers of phospholipids

with unsaturated fatty acyl chains. On the other hand, it helps to modulate membrane fluidity

and permeability of bilayers containing saturated phospholipids and sphingolipids (23).

Cholesterol also plays an important role in the production of all steroid hormones, bile acids,

and vitamin D as well as in cellular functions such as transmembrane signaling processes,

membrane trafficking and cell proliferation (24, 25). Nevertheless, insufficient or excessive

levels of cellular cholesterol can result in abnormal cellular processes, and subsequently, lead

to serious diseases such as atherosclerosis and type II diabetes (26). It is no wonder that

cholesterol homeostasis is among the most intensely regulated processes in cell biology.

1.1.2 SREBPs: main regulators of cholesterol homeostasis

Cells have developed a tightly controlled manner to regulate cholesterol homeostasis

via membrane-bound transcription factors called sterol regulatory element binding proteins

(SREBPs). SREBPs are members of the basic helix-loop-helix leucine zipper (bHLH-Zip)

family of transcription factors that regulate genes encoding proteins required for cholesterol,

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fatty acid, and phospholipid synthesis (28, 29, 30). SREBPs consist of three isoforms:

SREBP-1a, SREBP-1c and SREBP-2. SREBP-1a and SREBP-1c are products of the same

gene Srebp-1, but differ in their first exons by the alternative transcriptional start site.

SREBP-2 is the product of a separate gene, Srebp-2. SREBP-1c mainly regulates fatty acid

synthesis and insulin-mediated glucose metabolism, whereas SREBP-2 is involved in

cholesterol synthesis. In contrast, SREBP-1a seems to activate genes in both biosynthetic

pathways (28, 30, 31).

SREBPs are synthesized as inactive precursors that attach to the ER membrane in a

hairpin fashion (32, 33). To exert their transcriptional activity, SREBPs must translocate

from the ER to the Golgi for the proteolytic processing that converts precursor membrane-

bound SREBPs into their soluble active forms. Following translation, SREBPs bind to

SREBP cleavage-activating protein (SCAP) that plays a major role in trafficking of SREBPs

by acting as an escort protein and sterol sensor (34). However, during sterol-rich conditions,

SREBPs are retained in the ER by the binding of SCAP to the insulin-induced gene 1

(INSIG-1). This ER anchor protein binds to the sterol-sensing domain of SCAP and prevents

the SCAP/SREBP complex from entering the coat protein complex II (COPII)-dependent

ER-Golgi trafficking (35, 36). When intracellular cholesterol levels are low, SCAP

undergoes a conformational change, allowing its dissociation from INSIG-1; subsequently,

resulting in the interaction with the COPII trafficking complex and the proper transport of

SREBPs to the Golgi. Here, SREBPs are cleaved at two sites by two membrane-bound

proteases, site-1 protease (S1P) encoded by MBTPS1 gene and site-2 protease (S2P) encoded

by MBTPS2 gene, and release the active NH2-terminal domains that translocate to the

nucleus and activate lipid metabolism genes, such as HMGCR and the LDLR (35, 36, 37)

(Figure 1.1).

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Figure 1.1. The SREBP pathway. Following synthesis in the RE, SREBP transcriptional factors are retained at the membrane as a complex with SCAP. Both of the N- and C-terminus of SREBPs point toward the cytoplasm to form a hairpin structure. In response to sterol depletion, SCAP serves as an escort protein that directs SREBPs to transport vesicles, and subsequently, to the Golgi. In the Golgi, SREBPs undergoes two steps of proteolysis mediated by S1P and S2P, Golgi-located proteases. S1P cleaves the luminal loop between two membrane-spanning helices of SREBPs, whereas S2P separates the N-terminal bHLH-Zip domain from the membrane-spanning region. This second cleavage releases soluble SREBPs that translocate to the nucleus and transcriptionally activate the expression of critical genes in lipid metabolism. Image taken from Brown and Goldstein (2009) (27).

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In addition to regulation at the processing level, SREBP activity can be controlled in

numerous other ways. For example, in the absence of sterols, SREBP-2 and SREBP-1c can

activate the expression of their own genes in a feed-forward manner via the sterol response

elements (SRE) located in their respective promoter regions (29). Nuclear SREBPs can also

be modified by ubiquitination, leading to their degradation by the 26S proteasome or by

SUMO-1 to repress transcriptional activity (38, 39). Recently, it has been shown that sirtuin

1 (SIRT1), a NAD+ -dependent deacetylase, can decrease the stability of these transcriptional

factors via deacetylation (40). In contrast, the acetylation of SREBP-1a and SREBP-1c by

the transcriptional activator, p300, stabilizes their structures, resulting in significant increases

of the expression of target genes (29).

1.2 LDLR and LDL uptake

1.2.1 LDLR

An important component of cholesterol homeostasis is the SREBP-2 – regulated cell

surface LDLR. LDLR serves as the principal receptor for cellular cholesterol uptake,

mediating the endocytic removal of cholesterol-containing particles (VLDL, VLDL remnants

and LDL) from the circulation (41, 42).

Domain organization of LDLR: First discovered by Brown and Goldstein in 1974, the mature

LDLR is a modular, trans-membrane glycoprotein of 839 amino acids (42). The amino

terminus of the receptor contains seven adjacent LDLR type-A (LA) modules that act in

combination to bind to various lipoproteins, including LDL and β-VLDL particles. Among

the LA repeats, LA3 to LA7 are responsible for binding to LDL (43, 44, 45). Immediately C-

terminal to these ligand-binding repeats of the LDLR is the epidermal growth factor (EGF)-

precursor homology domain, including two EGF-like modules (EGF-A and EGF-B),

7

followed by a YWTD β-propeller domain and a third EGF-like module (EGF-C). This

homologous region of the LDLR is involved in the release of bound lipoproteins at low pH

as well as the receptor-recycling to the cell surface. Several studies confirmed that despite

maintaining binding to β-VLDL and to a lesser extent, LDL, deletion of the entire EGF-

homology domain not only eliminates the acid-dependent lipoprotein release but also inhibits

the efficient receptor-recycling (46, 47, 48). The importance of this EGF precursor domain is

also further highlighted by the fact that more than 50% of human familial

hypercholesterolemia (FH) point mutations occur in this region (49).

Following the EGF homology domain, a region that is rich in serine as well as

threonine and undergoes O-linked glycosylation serves as a spacer isolating the functional

domains of the LDLR from the cell surface. The glycosylated region is followed by a

transmembrane segment and a 50-residue cytoplasmic tail, which plays a necessary role in

receptor localization into clathrin-coated pits as well as receptor endocytosis (50, 51).

LDLR processing: Aiming at adjusting the number of LDLRs to provide sufficient

cholesterol for metabolic needs without producing cholesterol over-accumulation, LDLR

expression is subjected to feedback regulation via SREBP-2 (42). After synthesis, the

120kDa precursor form of LDLR undergoes several folding processes in the ER (52) (Figure

1.2). The receptor-associated protein (RAP) functions not only as a chaperone for the correct

folding of N-terminal LA repeats but also as an inhibitor of the premature binding of co-

expressed ligands (53); whereas a second kind of chaperone proteins (called Boca in

Drosophila) is thought to bind the β-propeller domain in order to facilitate its proper folding

(54). Briefly, each LA repeat in the ligand-binding domain contains two loops connected by

three disulfide bonds, of which the second loop carries four highly conserved acidic residues

8

Figure 1.2. Cellular itinerary of the LDLR. (1) The precursor form of the LDLR is synthesized and subjected to several folding steps in the ER. (2) After being transported to the Golgi where it undergoes extensive glycosylation, the LDLR is translocated to the cell membrane as a mature functional form. (3) At the cell membrane, the LDLR binds to circulating lipoproteins, especially LDL containing the single copy of apoB. (4) Subsequently, the receptor-ligand complex is endocytosed into clathrin-coated pits, mediated by the adaptor protein ARH, and delivered to endosomes. (5) In early endosomes where the low pH environment triggers release of the bound lipoprotein particle, the receptor separates from the ligand and recycles back to the cell surface for another round, while the released lipoprotein proceeds to lysosomes where its cholesterol esters are hydrolyzed. Images borrowed from Beglova and Blacklow (2005)(58).

9

near the C-terminal end of the module. These acidic residues are required to maintain the

structure of cysteine-rich repeats through forming a calcium-binding site with the consensus

sequence (55, 56). In the absence of calcium, the structural integrity as well as the

lipoprotein-binding capacity of LA repeats is impaired (57). In addition to the ligand-binding

repeats, the EGF-like modules, including EGF-A, EGF-B, and EGF-C, also contain three

disulfide bonds and a calcium ion in their calcium-binding sites each (56). Finally, the LDLR

precursor is transported to the Golgi where it undergoes extensive O-linked glycosylation to

form the mature 160kDa receptor found at the cell surface (52).

1.2.2 LDLR-mediated LDL uptake pathway

LDL uptake pathway (Figure 1.2): Binding of the LDLR to circulating lipoproteins, including

LDL – the most important physiological ligand, occurs at the cell surface. The LDLR binds

LDL via a single copy of the 550kDa ApoB, which accounts for the 1:1 stoichiometry

between LDL and the LDLR (59, 60). In the ligand recognition of LDLR, the C-terminal

acidic residues on each LA repeat form a discrete patch of electron-negative surface that

interacts with positive charges on ApoB. Indeed, selective chemical modifications of

conserved basic residues on ApoB completely disrupted the LDLR binding to LDL (61, 62).

Upon ligand binding, the receptor-ligand complexes are endocytosed into clathrin-

coated pits (63). Endocytosis of the LDLR requires the participation of a NPXY sequence in

the cytoplasmic tail and an adaptor protein called autosomal recessive hypercholesterolemia

(ARH) in hepatocytes. ARH contains a PTB domain that can simultaneously bind the NPXY

sequence of LDLR and components of the endocytic machinery, including a canonical

clathrin-binding sequence LLDLE and a sequence recognized by the β2-adaptin subunit of

adaptor protein AP-2 (64). The complexes are then delivered to endosomes, where the low

10

pH as well as low calcium environment triggers the release of bound lipoprotein particles.

After this dissociation, the receptors recycle back to the cell surface while the released

lipoproteins proceed to lysosomes where its cholesterol esters are hydrolyzed (42, 65). The

LDLR makes one round trip every 10 minutes whether it is carrying bound lipoproteins or

not, and had a total of several hundred trips during its 20-hour lifespan (66).

Critical conformational rearrangements for lipoprotein release by the LDLR: Dissociation of

LDL particles from the LDLR at low pH results from the formation of two interfaces

described below. The primary interactions required for LDL release are the contacts between

LA4 and LA5 repeats with the β-propeller domain, in which the propeller domain acts as an

alternative intramolecular ligand for the central lipoprotein-binding repeats at acidic pH

instead of LDL. In this structure, the calcium-binding loops of both LA4 and LA5 dock side

by side to the six-bladed sheet of the β-propeller domain, providing this domain with 14

major residues to form an interface mediated by hydrophobic and charged interactions.

Among these residues, three histidine residues (H190, H562, and H586) serve as pH sensors

to promote closure of the receptor at acidic conditions (49, 56, 58).

Structural studies also revealed another interface related to release of LDL particles at

low pH. Along with the terminal ligand-binding repeat LA7, the EGF-A module forms a pH-

invariant scaffold, functioning as an anchor to limit central ligand-binding repeats to a

smaller conformation search space and to help them easily dock into the propeller domain.

By this way, the elbow-shaped scaffold facilitates the intracellular closure of LDLR at low

pH, and then the release of bound lipoprotein particles as well as the receptor-recycling to

the cell surface by converting the receptor from an open ligand-binding active conformation

to a closed ligand-binding inactive conformation (49, 58).

11

1.3 PCSK9: a new player in cholesterol metabolism

The PCSK9 story started in February 2003 when Nabil Seidah, a biochemist at the

Clinical Research Institute of Montreal, and his colleagues discovered a new member of the

proprotein convertase family called neural apoptosis regulated convertase 1 (NARC-1) as

well as its coding gene located on the short arm of chromosome 1 (67). Mutations in this

newly discovered gene, whose name was changed to PCSK9 later, were found in two French

families with hypercholesterolemia in whom previous investigations failed to identify

mutations in either LDLR or ApoB genes (68). Especially, Kara Maxwell and Jay Horton not

only identified PCSK9 as a novel SREBP-regulated gene in mice but also uncovered its role

in regulating cholesterol via a previously unknown pathway (4, 69, 70). Since then, PCSK9

has become the most promising target for several hundred studies related to

hypercholesterolemia and CHD.

1.3.1 Structure and processing of PCSK9

Structure: PCSK9 is the ninth known member of the proteinase K subfamily of subtilisin-

related serine endoproteases, which is predominantly expressed in liver, intestine and kidney

(71). PCSK9 mRNAs are also present in the developing brains (67). The human PCSK9

gene, which is located on chromosome 1p32.3, is about 22kb-long and consists of 12 exons

encoding a 692 amino acid glycoprotein (4, 5, 67).

Like other members of this family, PCSK9 shares the general structure that consists of

a signal sequence, followed by a prodomain (residues 31-152), a subtilisin-like catalytic

domain (residues 153-447) that contains a conserved triad of residues (Asp-186, His-226,

and Ser-386), and a variable C-terminal domain (residues 452-692) (72) (Figure 1.3). The

prodomain functions not only as a chaperone required for the correct folding of the catalytic

domain in the ER but also as an inhibitor of catalytic activity (67).

12

Figure 1.3. Structure of PCSK9. Like other members of the proprotein convertase family, PCSK9 consists of a 30-amino acid signal sequence, followed by a prodomain (residues 31-152), a subtilisin-like catalytic domain (residues 153-451), and a variable C-terminal domain (residues 452-692) that is rich in histidine and cysteine. The catalytic domain contains a conserved triad of residues, including aspartate-186, histidine-226, and serine-386. PCSK9 is cleaved after a non-basic residue, between residues 152 and 153 (FAQ152↓SIP), producing a 14-kDa prodomain and a 60-kDa fragment. Posttranscriptional modifications of PCSK9 in the Golgi are also shown, in which the yellow circle represents sulfation at tyrosine-38, the blue circles represent phosphorylation at serine-47 and serine-688, whereas the green circle symbolizes glycosylation at asparagine-533. Images modified from Horton, Cohen, and Hobbs (2009)(72).

13

Cellular itinerary of PCSK9: PCSK9 is synthesized as an inactive 74kDa precursor that

undergoes intramolecular cleavage at the boundary between the prodomain and the catalytic

domain in the ER. While the first seven known convertases are typically cleaved after a basic

residue, PCSK9 is cleaved after a non-basic residue, between residues 152 and 153

(FAQ152↓SIP), producing a 14-kDa prodomain and a 60-kDa fragment (5, 67, 73). However,

after cleavage, the separated prodomain remains associated with the catalytic/C-terminal

domains, and its last four amino acids occlude the catalytic triad (74, 75). Subsequently, this

noncovalent complex is transported to the Golgi where it goes through a series of

posttranslational modifications, including glycosylation, phosphorylation and tyrosine

sulfation (72). PCSK9 is then rapidly secreted from the liver as a stable complex with its

prosegment. Another difference between PCSK9 and other family members is that most

proprotein convertases undergo a second proteolytic processing within the prodomain to

generate active proteases, no evidence of the second cleavage as well as no other protein

substrates for PCSK9, except itself in an autocatalytic manner, has been yet identified.

Structural studies have shown that PCSK9 lacks a typical loop structure that is required for

the second cleavage event (74, 76, 77).

1.3.2 The crystal structure of PCSK9

Three separate crystal structures of apo-PCSK9 have recently been published,

revealing a tightly bound prodomain that makes the active site inaccessible to exogenous

substrates as well as a C-terminal domain with a previously unknown fold (74, 78, 79). The

PCSK9 prodomain comprises two α helices and a four-stranded antiparallel β sheet, of

which the β sheet forms the interface between the prodomain and the catalytic domain via

hydrophobic and electrostatic interactions. Particularly, the four C-terminal amino acids of

14

the prodomain (residues 149-152) bind the catalytic site as a β strand, in an analogous

fashion to an inhibitory peptide. Gln-152 forms hydrogen bonds with His-226 and occupies

the oxyanion hole located between the Ser-386 backbone nitrogen and the Asn-317 side

chain amide. Furthermore, extra stabilization of the inhibitory peptide in this position,

provided by a 14- amino acid extension at the N-terminus of the prodomain, further blocks

access to the catalytic triad (74, 79).

The central part of the catalytic domain is composed of a seven-stranded parallel β

sheet, flanked on both sides by α helices. Of note, the N-terminal α helix undergoes a

considerable conformational shift following autocatalytic cleavage of pro-PCSK9, moving

more than 25Å from Gln-152, and is found in a position similar to that observed in other

mature subtilases. This change in conformation is thought to be a consequence of

autocatalysis and necessary for secretion (78, 79). Similar to other proprotein convertases,

the two α helices are involved in the interface with the inhibitory prodomain. Three disulfide

bonds are also identified in this domain. Although minor differences can be observed in their

positions, resulting from differences in the surrounding amino acid residues, the catalytic

triad of PCSK9 is highly conserved and superimposable when compared to other subtilases.

Asn-137 forms the oxyanion hole that has been proposed to be critical for catalysis (79).

However, it should be noted that, different from other members, the substrate-binding groove

of PCSK9, which governs the substrate specificity, is mostly neutral in lieu of being

negatively charged (74).

The C-terminal cysteine-rich domain is mainly composed of three subdomains of

antiparallel β strands folded in a truncated jellyroll motif. Each subdomain is stabilized by

three internal disulfide bonds between the first and six cysteines, the second and fifth

15

cysteines, and the third and fourth cysteines cross-linking β-sheets (β1 – β6, β2 – β6, and β3

– β5, respectively). This domain is also rich in histidine residues, with the majority of them

cluster on a surface between subdomains 2 and 3 (78, 79).

1.3.3 Regulation of PCSK9

Several findings have connected PCSK9 to cholesterol metabolism and CHD risk. In

fact, a causative association was established between two nonsense mutations of PCSK9

(Y142X and C679X) and low plasma LDL-C levels. Individuals who carry these mutations

have 28% less LDL-C compared with non-carriers, which is accompanied by an astonishing

88% lower risk of developing heart diseases. Except from lower levels of total cholesterol,

mainly LDL-C and triglycerides, no other clinical phenotypes such as high-density

lipoprotein (HDL) cholesterol levels are identified in this population. Likewise, European

people who carry the R46L missense mutation in PCSK9 gene also exhibit significantly

lower LDL-C levels, which are associated with 47% reduction in CHD risk (8, 80). In

contrast, two gain-of-function alleles (S127R and F216L) that act in a dominant fashion are

implicated in autosomal dominant hypercholesterolemia (ADH) in French families. ADH is

an inherited disorder characterized by a selective increase of LDL-C levels in plasma and

leading to cardiovascular complications. Importantly, a gain-of function mutation Asp-374-

Tyr (D374Y) leads to significantly higher serum total cholesterol levels (even levels of total

cholesterol on treatment with statins), which is accompanied by 10-year earlier development

of premature CHD in British and Norwegian families when compared with typical

heterozygous FH patients with known mutations in LDLR (5, 68).

Because of its importance role in LDL metabolism, it seems clinically relevant to

measure circulating levels of PCSK9 in humans. Several studies have developed ELISA

16

assays to measure PCSK9 concentrations in human plasma, which vary over a very wide

range (from 33 ng/ml to 4 µg/ml) among normal and apparently healthy individuals (84, 85,

86). The dissimilar results between these assays are likely attributable to differences in

specificities among antibodies to bind plasma PCSK9 and the recombinant PCSK9 standards

used in ELISA. More recently, PCSK9 plasma levels have been shown to be mirror markers

of hepatic cholesterol metabolism, with increased levels following refeeding and furthermore

following a diurnal rhythm (75). Thus different methods of blood collection may at least

partially explain previous observations of PCSK9 variance in human plasma samples.

Like other genes involved in cholesterol metabolism such as LDLR, PCSK9 is also

regulated primarily at the transcriptional level by SREBPs (69, 70). SREBP binding sites,

such as SRE and Sp1, were characterized in the promoter of human, mouse, and rat PCSK9

genes. Although SREBP-1c has been suggested to be responsible for the increased PCSK9

expression in response to liver X receptor (LXR) agonists, SREBP-2 appears to play a more

central role under physiological conditions. Hepatic expression of PCSK9 was significantly

decreased by long-term fasting, which suppressed SREBP-2 activity, and was restored by re-

feeding, which activated SREBP-2 (81, 82, 83). It was shown that PCSK9 was also down-

regulated by fenofibrate, an agonist of the nuclear peroxisome proliferator-activated receptor

α (PPARα), and up-regulated by cholestyramine, the bile acid-binding resin that stimulates

the clearance of circulating LDL (76). The physiological importance of these regulators

requires further studies.

1.4 PCSK9-mediated LDLR degradation

Soon after its discovery, several studies demonstrated that PCSK9 regulates LDL-C

levels in human plasma by targeting the LDLR for degradation. Early studies showed that

17

adenovirus-mediated overexpression of PCSK9 in mice resulted in a dramatic increase in

circulating LDL particles, which was dependent on the LDLR since no effects on LDL levels

were observed in LDLR null mice (4, 5, 6). In converse experiments, PCSK9-/- mice

exhibited a 2.8-fold increase in liver LDLR levels compared with the wild-type animals,

which were associated with a significant hypocholesterolemia profile of VLDL and LDL

(87). Also, PCSK9 when added to the medium of culture cells such as human hepatoma cells

(HepG2 and HuH7) or human embryonic kidney cells (HEK-293) significantly reduced

LDLR levels (6, 88, 89). Particularly, the results of parabiosis studies in wild-type mice and

PCSK9 transgenic mice provided clear evidence that circulating PCSK9 could function to

degrade LDLR in liver. It was reported that gain-of-function mutations of PCSK9 decreased

cell surface levels of the LDLR by 23% and LDL internalization by 38% as compared with

wild-type PCSK9, whereas loss-of-function mutations resulted in a 16% increase of cell

surface LDLRs and a 35% increase of LDL internalization (90). Through directly interacting

with the LDLR, PCSK9 inhibits the receptor recycling and induces the redistribution of

LDLR to late endosomes/lysosomes for degradation. However, the precise mechanisms by

which this occurs have yet been fully defined (Figure 1.4).

1.4.1 Crystal structure of LDLR/PCSK9 complex

Wild type PCSK9: Biomedical studies showed that PCSK9-induced LDLR degradation

involved the binding of PCSK9 to the first repeat in the EGF-precursor homology domain of

the LDLR (91) (Figure 1.5). This binding to EGF-A module occurs with a 1:1 stoichiometry

at a Kd of 170-750 nM at the neutral pH of plasma (92, 93, 94), and appears to be calcium-

dependent because the EGF-A interaction was completely abolished by sequestration of

divalent cations using EDTA (91). In contrast to the binding of LDL particles to the LDLR,

18

Figure 1.4. Model of LDLR degradative pathway mediated by secreted PCSK9 in hepatic cells. Like the LDLR, PCSK9 is synthesized as an inactive precursor in the ER. After undergoing the autocatalytic cleavage, the separated prodomain remains associated with the catalytic/C-terminal domains, and its last four amino acids occlude the catalytic triad. Afterward, this noncovalent complex is transported to the Golgi where it goes through several posttranslational modifications, and rapidly secreted from liver. PCSK9 interacts directly with the LDLR on the cell surface, followed by the endocytosis of the complexes via the adaptor protein ARH. Different from LDL particles that release from the LDLR in early endosomes, PCSK9 still maintains the binding to the LDLR with substantially higher affinity, and then routes the receptor to lysosomes for degradation by an unknown mechanism. Images modified from Horton, Cohen, and Hobbs (2009)(72).

19

Figure 1.5. The LDLR-PCSK9 complex at acidic pH. PCSK9-mediated LDLR degradation involves the binding of PCSK9 to the first EGF-homology domain of the LDLR. The affinity between PCSK9 and the LDLR is significantly enhanced in the acidic environment of endosomes. Here, three major domains of PCSK9 are shown in different color, whereas the LDLR is shown in light blue and the EGF-A domain is shown in dark blue. In this structure, phenylalanine-379 serves as a critical residue at the center of the hydrophobic surface. Several hydrogen bonds, which include interactions between (PCSK9)Aspartate-238 and (EGF-A)Asparagine-295 as well as (PCSK9)Threonine-377 and (EGF-A)Asparagine-309, are important for the specificity of the structure. Essential salt bridges between such as (PCSK9)Serine-153 and (EGF-A)Aspartate-299 , (PCSK9)Arginine-194 and (EGF-A)Aspartate-310, and (PCSK9)Aspartate-374 and (EGF-A)Histidine-306 are also shown. Images modified from Kwon et al. (2008) (96).

20

under acidic pH conditions of endosomes, the affinity between PCSK9 and the LDLR is

substantially higher (up to 170-fold) (74, 79, 92). His-306 of the EGF-A domain serves as an

important residue to help increase the binding affinity toward PCSK9 by moving from >9Å

away, at neutral pH, to 4Å away from PCSK9 – Asp-374 at acidic pH, forming an

intermolecular salt bridge (95). Also, it has been proposed that the prodomain as well as the

histidine-rich region of the C-terminus of PCSK9 might be also responsible for the pH-

dependent increase in affinity toward the LDLR (96, 97, 98).

The binding interface between PCSK9 and EGF-A is >20Å from the catalytic site of

PCSK9, mainly containing residues 367-381. The prodomain and the C-terminal domain of

PCSK9 are not involved in the binding to the EGF-A, whereas PCSK9 mainly contacts the

N-terminal region of the EGF-A domain, but not the C-terminus. Key residues that

contribute to the hydrophobic interface between PCSK9 and EGF-A include Phe-379, which

is located at the center of this surface and makes a number of contacts to EGF-A, and Cys-

378, which contacts an important residue of EGF-A (Leu-318) for specific binding between

PCSK9 and the LDLR. Besides, other polar interactions surrounding the interface are also

important for the specificity of PCSK9 binding to the EGF-A domain such as a hydrogen

bond between PCSK9 – Asp-238 and EGF-A – Asn-295 or a salt bridge between PCSK9 –

Ser-153 to EGF-A – Asp-299 (96). The conformational shift of PCSK9 – Ser-153 after

autocatalysis, moving more than 25Å from Gln152 as mentioned above, is required for

forming a salt bridge to EGF-A – Asp-299, which accounts for the incompetence of

uncleaved PCSK9 in binding to the LDLR (79). Mutation of these critical contacts in either

PCSK9 or the LDLR completely decreased the binding affinity between PCSK9 and the

receptor (91). Most importantly, in the structure of this complex, Arg-194 of PCSK9 forms a

salt bridge with EGF-A – Asp-310, a calcium-coordinating residue in the EGF-A repeat (96).

21

The specificity of this interaction was highlighted by studies in which Asp-310 residue of

EGF-A or PCSK9 – Arg-194 was changed into Glutamine. These modifications significantly

reduced the binding of PCSK9 to the LDLR by >90% (91). Because Asp-310 is involved in

the salt bridge with Arg-194, it just contributes one of its side-chain oxygen atoms for the

calcium coordination. The other calcium ligands in the EGF-A module consist of the side-

chain oxygen from Glu-296; the carbonyl oxygens of Thr-294, Leu-311, and Gly-314; and a

water molecule, forming a classic pentagonal bipyramid. Besides, there is a seventh ligand,

the carbonyl oxygen of Cys-292 (96).

Gain-of-function mutation D374Y: Among natural mutations of PCSK9, D374Y was

characterized to be about 10-fold more active than wild-type PCSK9 in degrading LDLRs (7)

because of 5 to 30-fold greater affinity in binding to the LDLR when compared with the

wild-type PCSK9. At neutral pH, Tyr-374 in lieu of Asp-374 forms a hydrogen bond with

the backbone carbonyl atom of EGF-A – Cys-319, resulting in a considerable increase in

total buried surface at the interface due to packing of the aromatic side chain of PCSK9 –

Tyr-374 against EGF-A – Leu-318 (93). At acidic pH, the hydroxyl group of Tyr-374 is ~3Å

from EGF-A – His-306, forming a favorable hydrogen bond. These additional interactions

account for the enhanced affinity of PCSK9 – Tyr-374 toward the LDLR, thus providing a

molecular basis for the severe hypercholesterolemia associated with the gain-of-function

mutation D374Y in humans (96).

1.4.2 Molecular characterization of PCSK9-mediated LDLR degradation

Although many aspects of PCSK9 as well as PCSK9-mediated LDLR degradation

have not been completely understood, over the last decade the research interests surrounding

this protein have dramatically grown, and mechanistic along with molecular studies related

22

to it have gained significant achievements. Numerous elegant works have provided us deeper

insights into underlying mechanisms for PCSK9-mediated LDLR degradation.

PCSK9 internalization requires the LDLR: In hepatocytes, secreted PCSK9 or purified

PCSK9 added to culture medium directly contacts with the LDLR on the cell surface.

PCSK9 is then internalized into the cells as a complex with the LDLR, and traffics to

endosomes/lysosomes to direct the receptor for degradation. PCSK9 endocytosis was

mediated by the LDLR as LDLR deficiency in hepatocytes from LDLR-null mice as well as

RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis (7,

99). Moreover, McNutt et al. (95) showed that addition of the LDLR(H306Y) subfragment,

which abolished the interaction of purified PCSK9 and the LDLR on the cell surface due to

the increased binding affinity toward PCSK9, completely blocked uptake of PCSK9 in

HuH7 cells. In addition, several studies showed that PCSK9 also binds to very low-density

lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), also known as

low-density lipoprotein receptor-related protein 8 (LRP8), with significantly lower affinity

when compared to LDLR interaction. For example, PCSK9 failed to bind cells that expressed

VLDLRs after 2-hour treatment (91); however, overnight incubation resulted in an increased

association of PCSK9 to these cells (110). VLDLR and ApoER2 receptors are not highly

expressed in hepatic cells, and they also do not contain Leu-318 residue shown to be

important for PCSK9 interaction. When Leu-318 was introduced into the VLDLR, this

construct was able to bind to PCSK9 equally compared to the LDLR (91), thus confirming

the defined molecular basis for the specificity of binding to the LDLR EGF-A domain.

Clathrin-mediated endocytosis is necessary for PCSK9 to degrade LDLRs: Clathrin-

mediated endocytosis is the major route of entry for many cargos in cells. Clathrin also

participates in the delivery of proteins from the Golgi to the cell surface (100). RNA

23

interference-mediated knockdown of clathrin heavy chain significantly abolished PCSK9-

mediated LDLR degradation without affecting the enrichment of LDLRs on the cell surface

(101, 102). So, PCSK9 uses the same endocytic machinery that imports LDL to promote

LDLR degradation.

The catalytic activity of PCSK9 is not required for its function: Replacement of catalytic

histidine residue in PCSK9 by alanine completely abolished its autocatalytic cleavage, and

resulted in PCSK9 remaining sequestered in the ER. So, autocatalytic activity seems

necessary for PCSK9 proper folding as well as secretion. However, it is not required for

PCSK9-mediated LDLR degradation (103). To overcome the requirement of PCSK9

catalytic activity for its maturation, a PCSK9 construct lacking its prodomain and containing

an inactive catalytic residue (Ser-386-Ala) was expressed in trans with the prodomain, which

yielded the catalytically inactive PCSK9 secreted in a manner similar to wild-type PCSK9.

Interestingly, this catalytic-dead version of PCSK9 degraded LDLRs to the same extent as a

catalytically active counterpart in HepG2 cells.

PCSK9 directs the LDLR to lysosome for degradation via a mechanism that is not related to

ubiquitination as well as proteasomes, autophagy, and canonical ESCRT pathway: Cell

fractionation and imaging studies confirmed that PCSK9 interfered with the LDLR recycling

to the cell surface, and routed the receptor to lysosomes for degradation. Moreover, treatment

of HuH7 cells with the lysosomal protease inhibitor E64d completely inhibited PCSK9-

mediated LDLR degradation (7, 102, 101). Some cell surface receptors, including the LDLR,

also undergo degradation via ligand-induced ubiquitination of lysine residues in their

cytoplasmic tails. These ubiquitinated receptors are then targeted to lysosomes or

proteasomes for degradation. IDOL is an E3 ubiquitin ligase that is transcriptionally

activated by LXR agonists, instead of SREBP transcriptional factors. Several studies showed

24

that by promoting the ubiquitination of the LDLR, IDOL targeted the receptor for lysosomal

degradation (13). However, mutation of lysine and cysteine residues in the cytoplasmic tail

of the LDLR failed to interrupt PCSK9-mediated receptor degradation, suggesting that

PCSK9 did not promote the LDLR ubiquitination. Besides, using proteasome inhibitors did

not affect the ability of PCSK9 to reduce LDLR levels in hepatic cells. So, PCSK9 acts

independently of ubiquitination and proteasomes to degrade LDLRs (102).

Several integral membrane proteins such as EGF receptor and ATP-binding cassette

transporter ABCA1 are delivered from endosomes to the multivesicular bodies (MVBs) and

then lysosomes by the endosomal sorting complex required for trafficking (ESCRT)

pathway. Inactivation of the initial components of the ESCRT machinery such as hepatocyte

growth factor-regulated Tyr-kinase substrate (HRS) and tumor susceptibility gene 101

(TSG101) did not inhibit PCSK9-mediated LDLR degradation in many cell types. However,

it is still possible that silencing RNA-mediated knockdown of HRS and TSG101 do not

completely inactivate the ESCRT pathway. Furthermore, PCSK9-induced LDLR degradation

may not require first components to enter the ESCRT pathway (102). These results still need

to be confirmed in future studies. Finally, depletion of Atg5 and Atg7, core components

required for autophagosome formation, failed to abolish LDLR degradation mediated by

PCSK9, indicating that PCSK9 did not use the basic molecular machinery of autophagy to

degrade LDLRs.

Structural requirements for PCSK9-mediated LDLR degradation: As mentioned above,

PCSK9 binds specifically to the EGF-A module of the LDLR in a calcium-dependent

manner (91). While other regions, including the β-propeller domain and at least three ligand-

binding repeats, are not required for PCSK9 binding or LDLR internalization, they play

25

important roles in PCSK9-mediated LDLR degradation. Similarly, the C-terminal domain of

PCSK9 is also essential for its activity on LDLRs although this domain does not bind the

receptor. Actually, it was suggested that the C-terminus might either prevent the LDLR from

binding to proteins required for recycling to the cell surface or provide a site for interaction

with other proteins that direct the receptor to lysosomes for degradation (104, 105). Further

studies are needed to define the functional role of these domains in PCSK9-mediated LDLR

degradation.

PCSK9 disrupts the acid-dependent change in LDLR conformation: Under acidic conditions

of endosomes, the LDLR changes from an open ligand-binding active conformation to a

closed ligand-binding inactive conformation, which facilitates the release of LDL particles

(49, 58). PCSK9 was shown to prevent the pH-dependent change in LDLR conformation;

however, disrupting this conformation shift of the receptor was not sufficient for explaining

PCSK9 activity since the LA4 and LA5 repeats were not required for PCSK9-mediated

LDLR degradation. Besides, PCSK9 efficiently degraded the mutant LDLR lacking four

ligand-binding repeats, but not the LDLR lacking five ligand-binding repeats although both

forms disrupted the pH-dependent conformational change of the LDLR (104).

1.4.3 Sites of action

Although the intracellular itineraries of PCSK9 and the LDLR are similar, their paths

become diverged at the cell surface, where the LDLR still attaches to the cell membrane and

PCSK9 is rapidly secreted into the circulation (58, 72). Secreted PCSK9 from transgenic

mice was able to reduce hepatic LDLR levels in recipient wild-type mice when it was

transferred via shared circulation in parabiosis experiments (7). In addition, single injection

or continuous infusion of recombinant human PCSK9 (32µg) to mice decreased hepatic

26

LDLR levels by ~90% within 60 minutes or 2 hours, respectively (106), supporting the

extracellular mechanism of PCSK9-mediated LDLR degradation. In this pathway, PCSK9 is

secreted from cells and directly interacts with the LDLR on the cell surface; subsequently,

extracellular PCSK9 is internalized together with the LDLR and directs the receptor for

lysosomal degradation. The extracellular mechanism requires the presence of ARH, an

adaptor protein necessary for the endocytosis process of the LDLR in hepatocytes. In fact,

PCSK9 failed to decrease surface LDLR levels, and the LDLRs were visualized almost

entirely on the cell surface in hepatocyes derived from Arh-/- mice (7, 99). However, a

previous study demonstrated that adenovirus-mediated PCSK9 overexpression promoted

similar hepatic LDLR degradation in ARH knockout and control mice (6). Additionally,

PCSK9 enhanced degradation of mature LDLRs as well as precursor forms of the LDLR in

post-ER compartments of HepG2 cells (107), indicating that PCSK9 might also act on the

LDLR before it reached the cell surface. Support for an intracellular mechanism is also

derived from a study in which Nassoury et al. identified the ER-localized proform of PCSK9

bound to the LDLR in the early secretory pathway when both proteins were overexpressed in

cultured cells (101). This may involve other domains of the LDLR since, as mentioned

previously, structural studies strongly support that the interaction of PCSK9 with the EGF-A

domain requires autocatalytic cleavage in order to create the binding interface of PCSK9.

Moreover, present data from HepG2 cells and mouse primary hepatocytes preferred a model

in which, depending on incubation time and concentrations, endogenous PCSK9 promoted

both extra- and intracellular degradation of the LDLR (108). Nevertheless, around the same

time, by addition of LDLR(H306Y) subfragments, which had increased binding affinity

toward PCSK9, to block the extracellular pathway, McNutt et al. recovered LDLRs in cells

overexpressing PCSK9 to levels approximating to those of control cells. They concluded that

27

if the intracellular pathway existed, it could only play a minor role in LDLR degradation

promoted by PCSK9 (95). However, further studies are still needed to clearly address the

relative contributions of intra- and extracellular mechanisms to PCSK9-mediated LDLR

degradation under both physiological and pathological conditions.

1.5 Research objectives

PCSK9 mainly acts as a secreted protein, and circulating PCSK9 can mediate

degradation of LDLRs in the liver. However, it is not clear to what extent PCSK9 affects

LDLRs in tissues other than the liver. Therefore, our objective for the first part of this study

was to assess PCSK9 activity on LDLR levels of hepatic and fibroblast cell lines. Studies in

mice demonstrated that although PCSK9 in plasma was capable of decreasing hepatic

LDLRs, no changes in LDLR levels were observed in the adrenal glands, proposing a cell-

type specific manner of PCSK9 activity (106). Moreover, Lagace et al. (2006) showed that

exogenous PCSK9 failed to degrade LDLRs in mouse embryonic fibroblasts despite its

normal LDLR-dependent uptake into these cells (7). Recently, it was also found that stable

levels of LDLR were similar in the brains of wild type, PCSK9-knockout and human

PCSK9-overexpressing mice (117). Based on these data, we hypothesized that PCSK9

degrades LDLR in a cell-type dependent manner.

In the second part of this study, our objective was to better understand the underlying

mechanisms for dissimilar responses to PCSK9-mediated LDLR degradation in hepatic and

fibroblast cells. Particularly, we wanted to gain further insights into the trafficking of PCSK9

after being internalized in responsive and nonresponsive cells. In PCSK9-responsive cells,

exogenous PCSK9 was endocytosed as a complex with the LDLR, followed by the delivery

of whole LDLR/PCSK9 complex to lysosome for degradation. However, it was shown that

PCSK9 failed to decrease LDLR levels in PCSK9-nonresponsive cells despite normal

28

cellular uptake; however, the fate of PCSK9 after binding to the LDLR in these cells was still

unknown. Illuminating the fate of PCSK9 in responsive and nonresponsive cells allowed us

to explore potential factors that might affect PCSK9-induced LDLR degradation in these cell

types.

To summarize, understanding how fibroblast cells are resistant to PCSK9-mediated

LDLR degradation will help us gain important information relevant to the elucidation of

molecular mechanisms by which PCSK9 degrades LDLRs as well as to the development of

PCSK9 inhibitors for treatment of CHD.

29

2. Materials and Methods

2.1 Materials

2.1.1 Chemicals and reagents

Cell culture medium DMEM, fetal bovine serum (FBS), newborn calf serum, and

human transferrin were obtained from Gibco – Life Technologies. LysoTracker® Red DND-

99, AlexaFluor® 488 protein labeling kit, and AlexaFluor® 647 human transferrin were

purchased from Molecular Probes – Life Technologies. We also obtained E-64 N-[N-(L-3-

trans-carboxyoxirane-2-carbonyl)-L-leucyl]-agmatine and EDTA-free CompleteTM Protease

Inhibitor Tablets from Roche; EZ-LinkTM Sulfo-NHS-SS-Biotin from Thermo Scientific;

Lipofectamine 2000 from Invitrogen – Life Technologies; PureProteomeTM Streptavidin

Magnetic Beads from Millipore; and IRDye® 800CW Streptavidin from LI-COR

Biosciences. All other chemicals and reagents were obtained from Sigma unless otherwise

specified.

2.1.2 Antibodies

Monoclonal anti-LDLR antibody C7 was purified by Protein G affinity

chromatography from conditioned medium of cultured mouse hybridomas purchased from

Cerdalane Laboratories (ACTT); rabbit anti-serum 3143 against the C-terminal 14 amino

acids of LDLR was the kind gift of Joachim Herz (University of Texas Southwestern

Medical Center, Dallas, TX); a mouse antihuman transferrin receptor (mbv) antibody was

purchased from Life Technologies; monoclonal anti-actin antibody (AC-10) and monoclonal

anti-FLAG M2 antibody were from Sigma-Aldrich. Secondary IRDye-labeled goat anti-

mouse and anti-rabbit IgG antibodies were from LI-COR Biosciences.

30

2.2 Protein assay

Protein assay was performed using PierceTM BCA Protein Assay Kit (Thermo

Scientific) according to the manufacturer’s instructions. BCA Reagent A consists of sodium

carbonate, sodium bicarbonate, bicinchoninic acid and sodium tartrate in 0.1M sodium

hydroxide, whereas BCA reagent B contains 4% cupric sulfate. Reagent B was mixed with

reagent A in a 1:50 ratio to prepare the BCA working reagent, which is stable as a clear and

green solution. The sample was diluted with ddH2O to a final volume of 50 µl, in which the

sample volume depends on its predicted concentration. Standard curves were prepared using

a bovine albumin serum (BSA) reagent with concentrations ranging from 4-32 µg/ml. 1 ml

of the BCA working reagent was added to all samples that were then incubated at 37oC for

30 minutes. Absorbance was read at 562 nm on a BIOWAVE II (Biochrom Ltd.) and plotted

against the determined standard curve.

2.3 Purification of human wild-type PCSK9 and PCSK9(D374Y)-FLAG fusion

proteins

FLAG epitope-tagged recombinant human wild-type PCSK9 and PCSK9 containing

the gain-of-function D374Y mutation were produced in stably transfected HEK293S cells

and purified similar to previously described (7). Briefly, HEK293S cells stably expressing

FLAG-tagged wild-type PCSK9 or PCSK9-D374Y were cultured in suspension without CO2

in UltraDOMATM hybridoma serum-free growth medium (Lonza) supplement with 10%

(v/v) FBS, 2 mM L-Glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate.

Recombinant PCSK9 was purified from the conditioned medium of HEK293S by using anti-

FLAG M2 affinity gel (Sigma) chromatography according to the manufacturer’s instructions,

followed by size-exclusion chromatography on a Tricorn high performance column

31

(Superdex 200 10/300 GL-GE Healthcare). Fractions containing PCSK9 peak were

concentrated approximately 8-fold using Amicon Ultra-4 Centrifugal Filter Units (10 kDa-

molecular weight cut-off) (Millipore). Protein concentration was identified by protein assay,

and protein purity was monitored by SDS-PAGE along with Coomassie Brilliant Blue R-250

Staining (Bio-Rad).

2.4 Tissue culture medium

Medium A contained DMEM and 4.5 g/l glucose, supplemented with 100 U/ml

penicillin and 100 µg/ml streptomycin sulfate. Medium B contained medium A

supplemented with 10% (v/v) FBS. Medium C contained medium A with 5% (v/v) newborn

calf lipoprotein-deficient serum (NCLPDS), 50 µM sodium mevalonate, and 10 µM

pravastatin. Medium D contained medium A with 5% (v/v) NCLPDS, 1 µg/ml 25-

hydroxycholesterol, and 10 µg/ml cholesterol. Medium E contained DMEM and 1 g/l

glucose, supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin sulfate.

Medium F contained medium E supplemented with 10% (v/v) FBS. Medium G contained

medium E with 5% (v/v) NCLPDS, 50 µM sodium mevalonate, and 10 µM pravastatin.

Medium H contained medium E with 5% (v/v) NCLPDS, 1 µg/ml 25-hydroxycholesterol,

and 10 µg/ml cholesterol.

2.5 Protein labeling

2.5.1 AlexaFluor® 488- labeled proteins

Recombinant human wild-type PCSK9 along with PCSK9-D374Y, and C7 antibody

were labeled according to the manufacturer’s instructions. Briefly, the protein was diluted to

2 mg/ml in HEPES-buffered saline buffer, supplemented with 2 mM CaCl2 (HBS-C), and

was subsequently mixed with 50 µl of 1M bicarbonate. The protein solution was transferred

32

to the vial of reactive dye that contained a magnetic stir bar, and was suspended a few times

to completely dissolve the dye. The reaction mixture was allowed to sit for 1 hour at room

temperature, followed by the separation of labeled proteins from unincorporated dye using

size-exclusion chromatography. Protein concentration was identified by protein assay, and

labeled proteins were stored in aliquots at -80oC.

2.5.2 Biotin-labeled proteins

Recombinant human wild-type PCSK9 as well as PCSK9-D374Y, and human

transferrin were diluted to 2 mg/ml in HBS-C buffer. 40 µl of 0.67M borate buffer was added

to each protein solution to get 50 mM final concentration. A 10 mM solution of Sulfo-NHS-

SS-Biotin was prepared by dissolving 6mg of reagent in 1 ml of ddH2O. The appropriate

volume of biotin solution added to each protein solution was calculated according to the

manufacturer’s instructions. The reactions were incubated on ice for 2 hours; subsequently,

was stopped by quenching buffer (25 mM Tris-HCl, pH 7.4; 192 mM Glycine). Labeled

proteins were purified by size-exclusion chromatography, and protein concentration was

determined by protein assay.

2.6 PCSK9 cellular uptake assay

SV589 human skin fibroblasts were grown in medium B to ~80% confluence, and then

were cultured in sterol-depleting medium C and sterol-supplemented medium D for 18 hours

to up-regulate and down-regulate LDLR expression, respectively. Similarly, HepG2 human

hepatoma cells were grown in low-glucose medium F to ~80% confluence, and then were

cultured in medium sterol-depleting G and sterol-supplemented medium H for 18 hours.

These mediums were supplemented with E64 to inhibit lysosomal degradation (102).

Following 1 hour of incubation with AlexaFluor® 488-labeled PCSK9-D374Y (1 µg/ml),

33

the medium was replaced with stripping buffer (100 mM Na 2-mercapto-ethanesulfonate; 50

mM Tris, pH 8.6; 100 mM NaCl; 1 mM EDTA; 0.2% BSA). Cells were collected and

filtered through a 70 µM sterile cell strainer (BD Biosciences). An additional 0.5 ml of sterile

phosphate-buffered saline (PBS) was used to collect excess cells from the wells. Samples

were spun at 1,000 rpm for 5 minutes and resuspended in 300 µl of PBS, followed by the

analysis on BD FACSAria flow cytometer and cell sorter (BD Biosicences).

2.7 Biotinylation and immunoblot analysis

SV589 cells were grown in medium B to ~80% confluence, and then were cultured

overnight in sterol-depleting medium C to induce LDLR expression prior to treatment with

purified wild-type PCSK9 or PCSK9-D374Y. Similarly, HepG2 cells were grown in medium

F to ~80% confluence, and then were cultured overnight in medium G prior to treatment with

PCSK9. The cells were incubated with PCSK9 for 6 hours, except the overnight

experiments, in which the cells were treated with PCSK9 for 18 hours.

Cell surface proteins were biotinylated as previously described (7). Whole cell extracts

were prepared with Tris lysis buffer (50 mM Tris-Cl, pH 7.4; 150 mM NaCl; 1% Nonidet P-

40 (EMD Biosciences); 0.5% sodium deoxycholate; 5 mM EDTA; 5 mM EGTA;

CompleteTM protease inhibitor cocktail; 1 mM phenylmethylsulfonyl fluoride (PMSF)).

Three quarters of each cell lysate was added to the mixture of Tris lysis buffer and 50 µl

streptavidin magnetic beads to bring the final volume to 500 µl. The mixture was rotated

overnight at 4oC. The pellets were then precipitated, and washed three times in lysis buffer.

Cell surface proteins were eluted from the beads by adding 1X SDS loading buffer (50 mM

Tris-HCl, pH 6.8; 1% SDS; 5% glycerol; 10 mM EDTA; 0.0032% bromophenol blue) and

incubating for 10 minutes at 96oC. Proteins were subjected to 8% SDS-PAGE and

34

transferred to nitrocellulose membranes (Bio-Rad) for immunoblot analysis. Secondary

infrared dye (IRDye800)-labeled antibodies were used for detection on a LI-COR Odyssey

infrared imaging system (LI-COR Biosciences). Band intensity was quantified using

Odyssey 2.0 software.

2.8 LDLR degradation assay on 917 and HuH7 cells

Similarly, 917 human foreskin fibroblasts and HuH7 human hepatoma cells were

cultured in medium B to ~80% confluence, and then were incubated in lipoprotein-deficient

medium C for 18 hours to up-regulate LDLR expression prior to treatment with purified

wild-type PCSK9 (5, 10, 20 µg/ml) or PCSK9-D374Y (2 µg/ml) for 6 hours. After PCSK9

treatment, cells were washed twice with PBS immunofluorescence buffer (PBS-IF; 10 mM

Na2HPO4, 225 mM NaCl, 2 mM MgCl2, 0.1 mM CaCl2, pH 7.4), and fixed with 3.7%

formaldehyde in PBS-IF for 15 minutes. Cells were then incubated for 5 minutes with 1%

glycine (w/v) in PBS-IF and rewashed three times with PBS-IF, followed by the standard

staining procedure as described. Briefly, cells were blocked with 1% BSA (w/v) in PBS-IF

for >30 minutes at room temperature prior to overnight incubation with the primary

antibody, AlexaFluor® 488-labeled C7 antibody. C7 antibody was used at 20 µg/ml to detect

cell surface LDLRs. At the final steps, cells were subjected to three 10-minute washes in 1%

BSA (w/v) in PBS-IF, and collected for flow cytometry as described above. Samples were

analyzed on BD FACSAria flow cytometer and cell sorter (BD Biosicences).

2.9 125I-radiolabeled PCSK9 degradation assay

SV589 cells were grown in medium B to ~80% confluence, and the medium was then

switched to medium C. Similarly, HepG2 cells were grown in medium F to ~80%

confluence, and the medium was then switched to medium G. After 18 hours, the cells were

35

washed with PBS and incubated in 1 ml of medium A containing ~5 µg/ml 125I-labeled wild-

type PCSK9 or ~1 µg/ml 125I-labeled PCSK9-D374Y for 1 hour. In negative control, 100 µM

chloroquine was added to culture medium 30 minutes prior to and during later incubation to

inhibit lysosomal degradation of internalized PCSK9. The medium was removed and the

cells were washed with ice-cold PBS for 5 minutes. The cells were then incubated in 1.5 ml

of medium A for 6 hours at 37oC. The amounts of 125I-mono-iodotyrosine as a catabolic

product when internalized 125I-labeled PCSK9 was degraded in lysosomes were determined

in the medium as previously described (104). Briefly, the medium was collected and spun at

3,000 rpm for 5 minutes to remove unattached cells. 1.3 ml of the medium was transferred to

new tubes, followed by the addition of 130 µl of 100% trichloroacetic acid (TCA) to obtain a

TCA final concentration of 10% (v/v). Samples were incubated on ice for more than 30

minutes and spun at 13,000 rpm for 15 minutes to precipitate residual 125I-labeled PCSK9

that had not been internalized and released from the cell surface into the medium. 1 ml of

supernatant was removed to 13x100mm glass tubes, and was mixed with 10 µl of 40% KI.

40 µl of 30% hydrogen peroxide was added, and the mixture was extensively vortexed.

Samples were allowed to stand for 10 minutes at room temperature. 2 ml of chloroform were

added, followed by 15-minute incubation at room temperature. These steps aimed at

removing any free iodide. Finally, sample tubes were spun at 2,500 rpm for 2 minutes, and

700 µl of the upper aqueous layer was removed to test tubes for γ counting. The amounts of

125I-mono-iodotyrosine were normalized to protein levels determined by protein assay, and

were corrected for negative controls.

36

2.10 Recycling assay

2.10.1 Transferrin

SV589 cells were cultured in medium B to ~80% confluence using 12-well plates, and

then were cultured in serum-free medium A for >1 hour to deplete endogenous transferrin

prior to incubation with labeled complexes. For monensin-treated wells, 50 µM monensin

was added to culture medium more than 1 hour prior to and during later incubation to inhibit

recycling of internalized Transferrin. Biotin-labeled transferrin was incubated with IRDye®

800CW Streptavidin in medium A for 1 hour at 37oC, and the complexes were then added to

the cells. After 1 hour, the cells were incubated with 20 mM Tris(2-carboxyethyl)phosphine

hydrochloride (TCEP) in buffer B (PBS, 0.1 mM CaCl2, 2 mM MgCl2, 0.5% BSA (w/v)) for

20 minutes at 4oC to remove cell surface labeled complexes before being subjected to two

10-minute washes with 5 mg/ml Iodoacetamide in buffer B. The cells were rewashed with

buffer B as well as PBS-CM (PBS, 0.1 mM CaCl2, 2 mM MgCl2), and then incubated in

medium A, supplemented with 20 mM TCEP, for 2 hours at 37oC. The plate was directly

scanned on the LI-COR system at the indicated time intervals to visualize internalized

transferrin. Signal intensity was quantified using Odyssey 2.0 software, corrected for

background using untreated wells, and normalized to DNA levels stained by DRAQ5TM

(Cerdalane Laboratories, Canada) (1:10,000).

2.10.2 PCSK9

SV589 cells were grown in medium B to ~80% confluence using 12-well plates, and

then were cultured in medium C, supplemented with 150 µM E-64, for 18 hours. The

recycling assay was performed as described above, with the exception that the internalized

complexes were chased for 6 hours in the continuous presence of both TCEP and E-64.

37

2.11 Live cell imaging for co-localization studies

2.11.1 PCSK9 and LysoTracker

SV589 cells were cultured in medium B to ~80% confluence using µ-slide 8-well

plates, and then were cultured in medium C1 (medium C supplemented with 150 µM E-64)

for 18 hours. HepG2 cells were seeded in medium F using µ-slide 8-well plates to 80%

confluence, and then were cultured overnight in medium G1 (medium G supplemented with

150 µM E-64). The medium was replaced with medium C1 (or G1) containing 30 µg/ml

AlexaFluor® 488-labeled wild-type PCSK9 or 5 µg/ml AlexaFluor® 488-labeled PCSK9-

D374Y, and the cells were incubated for 1 hour at 37oC. The cells were washed two times

with medium A before internalized AlexaFluor® 488-labeled PCSK9 was chased for 6 hours

at 37oC in medium C1 (or G1). LysoTracker® Red DND-99 was used as per the

manufacturer’s instructions. Briefly, the stock solution of 1mM probe was diluted to the final

working concentration (200 nM) in medium C1 (or G1). The cells were then incubated with

LysoTracker for 2 hours prior to being washed twice with medium A and directly observed

on a microscope at the indicated time intervals. Images were taken using the confocal

microscope Fluoview FV1000 version 2.1 (Olympus), in which PCSK9 was visualized using

a 488nm laser, and a 543nm laser was used for LysoTracker. The percentage of co-

localization was quantified using Image J (http://rsb.info.nih.gov/ij/), and corrected for

background using negative images.

2.11.2 PCSK9 and transferrin

SV589 cells were grown in medium B to 80% confluence using µ-slide 8-well plates,

and then were cultured in medium C1 for 18 hours. Following >1 hour incubation in serum-

free medium A1 (medium A supplemented with 150 µM E-64) to deplete endogenous

38

transferrin, cells were treated with AlexaFluor® 488-labeled PCSK9-D374Y (or

AlexaFluor® 488-labeled wild-type PCSK9) and AlexaFluor® 647-labeled transferrin for 1

hour at 37oC. The cells were then washed two times with PBS-IF, and chased for 2 hour in

medium A1. Images were directly taken on the confocal microscope Fluoview FV1000

version 2.1 (Olympus) using the 488nm laser for PCSK9 and a 633nm laser for transferrin.

2.11.3 Transferrin and Rab4 (RAS-related GTP-binding protein 4)

SV589 cells were cultured in medium B using glass bottom dishes. When reaching

60% confluence, cells were transiently transfected with N-terminal tRFP-tagged Rab4

expression plasmid (OriGene Technologies) using Lipofectamine 2000 according to the

manufacturer’s protocols. After 20 to 24 hours, the cells were incubated in serum-free

medium A for >1 hour, followed by the addition of 100 µg/ml AlexaFluor® 647-labeled

transferrin for 1 hour at 37oC. The cells were then washed two times with PBS-IF, and

chased for 2 hour in medium A. Images were taken using the confocal microscope Fluoview

FV1000 version 2.1 (Olympus), in which Rab4 was visualized using the 543nm laser, and

the 633nm laser was used for Transferrin.

2.12 Mutagenesis

A pCMV4 vector that contains the full-length sequence encoding human LDLR

(pLDLR17) was used as a template for mutagenesis. Mutagenesis was carried out using a

modified protocol of QuickChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA)

(109). Briefly, primers were designed to contain extended non-overlapping sequences at the

3’ end, which has a melting temperature (Tm no) 5 to 10oC higher than that of primer-primer

complementary sequences at the 5’ end (Tm pp). The mutation sites could be placed either in

the overlapping region or the complementary region. A 50 µl PCR reaction consists of 50 ng

39

templates, 0.3 µM each primer, 0.3 mM dNTPs and 1 unit Phusion DNA polymerase (New

England Biolabs). The PCR cycles were initiated at 95oC for 5 minutes, followed by 25

amplification cycles. Each cycle includes 95oC for 1 minute, 76oC for 1 minute and 72oC for

15 minutes. The cycles were finished with an annealing step at 75oC for 1 minute and an

extension step at 72oC for 30 minutes. The following steps were performed according to the

instructions of the QuickChangeTM site-directed mutagenesis kit.

2.13 Transient transfection

SV589 cells were grown in 100-mm dishes using medium B. HepG2 cells were grown

in 60-mm dishes using medium F. When reaching ~70% confluence, cells were transfected

with wild-type LDLR, LDLR(E296Q) or LDLR(EGF66) expression plasmids using

Lipofectamine 2000 as per the manufacturer’s protocols. After 5 hours, cells were incubated

overnight in sterol-supplemented medium D to suppress endogenous LDLR expression prior

to treatment with wild-type PCSK9 or PCSK9-D374Y for 6 hours. Cell surface proteins were

isolated by biotinylation as previously described (7). Whole cell extracts as well as cell

surface proteins were prepared for SDS-PAGE and immunoblot analysis as mentioned

above.

2.14 Statistical analysis

All presented values are mean ± standard deviation. Statistical analysis was determined

by Student’s t-test and GraphPad Prism 5 software.

40

3. Results

3.1 Exogenous PCSK9 significantly decreased LDLR levels in HepG2 hepatic cells, not

in SV589 fibroblast cells

3.1.1 PCSK9 at physiological concentrations

Although it caused robust degradation of membrane hepatic LDLRs during continuous

infusion into wild-type mice, purified PCSK9 had minimal effects on cell surface LDLRs in

the adrenals (106), suggesting that PCSK9-mediated LDLR degradation was cell-type

specific. To test this hypothesis, we assessed the effects of exogenous added PCSK9 on

LDLR levels of HepG2 hepatic cells and SV589 fibroblast cells.

Both of these cell types were incubated in sterol-depleting medium containing statin

(pravastatin) for 18 hours before PCSK9 treatment to up-regulate LDLR expression.

Recombinant purified PCSK9, including wild-type PCSK9 and the mutant PCSK9-D374Y,

was added to the medium of cultured cells at physiological concentrations and incubated for

6 hours. As discussed in the introduction, PCSK9 concentrations in human plasma range

from 33 ng/ml to 4 µg/ml among healthy individuals (84, 85, 86). Therefore, we treated the

cells with wild-type PCSK9 at 2.5 µg/ml and 5 µg/ml, whereas PCSK9-D374Y was used at

10-fold lesser concentration because this mutant has been characterized to be about 10-fold

more active than wild-type PCSK9 in degrading LDLRs as mentioned above (7).

Immunoblot analyses of whole cell proteins as well as cell surface proteins showed that

in response to PCSK9 addition, LDLR levels didn't significantly change in SV589 fibroblast

cells despite a dramatic decrease of LDLRs in HepG2 hepatic cells, compared to control

untreated cells (Figure 3.1). Particularly, cell surface LDLRs became nearly undetectable

41

Figure 3.1. Addition of PCSK9 at physiologically relevant concentrations causes robust LDLR degradation in HepG2 human hepatoma cells, but not SV589 human fibroblast cells. (A) HepG2 cells were cultured to ~80% confluence in medium F. The medium was then switched to sterol-depleting medium G, and the cells were incubated for 18 hours to induce LDLR expression. Wild-type PCSK9 at 2.5 µg/ml and 5 µg/ml along with PCSK9-D374 at 10-fold lesser concentrations were added to the medium for 6 hours. Cell surface proteins were isolated by biotinylation as described in Materials and Methods. Whole cell lysates and streptavidin-precipitated proteins were subjected to 8% SDS-PAGE followed by immunoblot analysis of LDLR, PCSK9 and TFR. Secondary infrared dye (IRDye800)-labeled antibodies were used for detection on the LI-COR Odyssey infrared imaging system. (B) SV589 cells were cultured to ~80% confluence in medium B, and then were incubated in sterol-depleting medium C for 18 hours to induce LDLR expression. SV589 cells were treated with PCSK9 and whole cell proteins as well as cell surface proteins were analyzed as described in Panel A. All experiments were performed twice with similar results.

42

when HepG2 cells were incubated with either 5 µg/ml of wild-type PCSK9 (lane 10) or 0.5

µg/ml of PCSK9-D374Y (lane 8), while whole cell LDLRs were decreased in a lesser extent

(lane 2-5). Nevertheless, although there was a minor reduction of membrane LDLRs resulted

from PCSK9 treatment (lane 7-10), no differences were observed in whole cell LDLRs of

SV589 fibroblast cells (lane 2-5), indicating that a portion of cell surface LDLRs might be

internalized, but not degraded as well as not recycled yet.

Exogenous PCSK9 was detected in a concentration-dependent manner in whole cell

extracts (lane 2-5), whereas no PCSK9 was observed in cell surface proteins. Therefore,

most of the cell-associated PCSK9 was internalized normally in both HepG2 hepatic cells

and SV589 fibroblast cells. Levels of internalized PCSK9-D374Y were significantly higher

in these cells when being compared with wild-type PCSK9 perhaps due to its 5 to 30-fold

greater affinity in binding to the LDLR.

3.1.2 PCSK9 at higher concentrations

Importantly, when purified PCSK9 was added to the medium at higher concentrations

compared with physiological levels, SV589 fibroblast cells were still resistant to PCSK9-

mediated LDLR degradation (Figure 3.2). Wild-type PCSK9 at 5 µg/ml or 10 µg/ml didn’t

considerably affect the number of both whole cell (lane 3-4) and cell surface (lane 8-9)

LDLRs in SV589 fibroblast cells. When the dose was increased up to 20 µg/ml of wild-type

PCSK9, whole cell LDLRs fell by ~20% compared to the control (lane 5), but it should be

noted that this concentration is remarkably higher than the physiological levels of PCSK9 in

human plasma, which range from 33 ng/ml to 4 µg/ml (84, 85, 86). Notably, PCSK9-D374Y

at 2 µg/ml dramatically reduced LDLR levels on the cell surface (lane 7) despite no changes

43

Figure 3.2. Changes of endogenous LDLR levels in SV589 human fibroblast cells when PCSK9 was added at higher concentrations. SV589 cells were grown in medium B to ~80% confluence, and then were cultured overnight in lipoprotein–deficient medium C to induce LDLR expression. Wild-type PCSK9 (5, 10, and 20 µg/ml) and PCSK9-D374Y (2 µg/ml) were added to the medium for 6 hours. Cell surface proteins were biotinylated as described in Materials and Methods. Whole cell lysates and membrane proteins were subjected to 8% SDS-PAGE followed by immunoblot analysis of LDLR, PCSK9 and TFR. Secondary detection used infrared dye (IRDye800)-labeled antibodies. Blots were visualized and quantified using the LI-COR Odyssey infrared imaging system. LDLR levels were normalized to TFR expression and expressed relative to control cells (no addition – NA). Graphs represent the means ± standard deviation from three independent experiments. * indicates a statistical difference between columns with significance p <0.05 by Student’s t-test; ** p<0.005.

44

in the levels of whole cell LDLRs (lane 2), therefore again, suggesting that LDLR might be

rapidly taken up into the cells, but not degraded or recycled.

In contrast, higher doses of exogenous PCSK9 resulted in a more significant reduction

of LDLRs in HepG2 hepatic cells (Figure 3.3). Especially, whole cell as well as cell surface

LDLRs were decreased in a PCSK9 concentration-dependent manner. Incubation of HepG2

cells for 6 hours with 5 µg/ml wild-type PCSK9 degraded LDLR levels by approximately

50% for whole cell proteins (lane 3), and by approximately 80% for cell surface proteins

(lane 8). Especially, it was hard to detect LDLRs in both whole cell and cell surface proteins

when the cells were incubated with 20 µg/ml wild-type PCSK9 (lane 5 and lane 10) or 2

µg/ml PCSK9-D374Y (lane 2 and lane 7), in which the mutant at 2 µg/ml was as effective as

20 µg/ml of wild-type PCSK9 in reducing LDLR levels.

3.2 Long incubation time did not interfere with the resistance of SV589 fibroblast cells

to PCSK9-mediated LDLR degradation

A report demonstrated that upon 2-hour incubation, PCSK9 failed to bind cells that

expressed VLDLRs, a close ortholog of the LDLR (91); whereas a recent study showed that

overnight incubation with purified added PCSK9 led to an increased association of PCSK9 to

these cells, and subsequently, an enhanced degradation of VLDLRs (110). Therefore, to

confirm that PCSK9 has minimal effects on LDLR levels of SV589 fibroblasts and to

eliminate the contribution of different uptake time to the observed resistance of SV589

fibroblast cells to PCSK9-mediated LDLR degradation, we decided to increase incubation

time with both of wild-type PCSK9 and PCSK9-D374Y.

SV589 and HepG2 cells were cultured as described in the above experiment, with the

exception that these cells were incubated with PCSK9 for 18 hours. In HepG2 cells, overnight

45

Figure 3.3. Changes of endogenous LDLR levels in HepG2 human hepatoma cells when PCSK9 was added at higher concentrations. HepG2 cells were grown in medium F to ~80% confluence, and then were cultured in sterol–depleted medium G for 18 hours to induce LDLR expression. Wild-type PCSK9 (5, 10, and 20 µg/ml) and PCSK9-D374Y (2 µg/ml) were added to the medium for 6 hours. Cell surface proteins were biotinylated as described in Materials and Methods. Whole cell lysates and membrane proteins were subjected to 8% SDS-PAGE followed by immunoblot analysis of LDLR, PCSK9 and TFR. Secondary detection used infrared dye (IRDye800)-labeled antibodies. Blots were visualized and quantified using the LI-COR Odyssey infrared imaging system. LDLR levels were normalized to TFR expression and expressed relative to control cells (no addition – NA). Graphs represent the means ± standard deviation from three independent experiments. ** indicates a statistical difference between columns with significance p <0.005 by Student’s t-test.

46

treatment with exogenous purified PCSK9 made cell surface as well as whole cell LDLR

levels became undetectable (lane 2-5 and 6-10), even with 5 µg/ml of wild-type PCSK9

(Figure 3.4). However, SV589 fibroblasts were still highly resistant to PCSK9 upon

overnight treatment. The number of LDLRs in cells incubated with 20 µg/ml wild-type

PCSK9 was similar to those in untreated cells (lane 5). LDLRs significantly decreased in

response to overnight treatment with 2 µg/ml PCSK9-D374Y for cell surface proteins (lane

7), and in a lesser extent for whole cell proteins (lane 2).

Internalized wild-type PCSK9 was also detected in a concentration-dependent manner

(lane 2-5) while PCSK9-D374Y at 2 µg/ml was taken up into cells as effectively as 20 µg/ml

wild-type PCSK9.

3.3 PCSK9 was ineffective to degrade LDLRs in another fibroblast cell line, 917

foreskin fibroblasts

To further confirm that PCSK9 significantly down-regulated the number of LDLRs in

hepatic cells while it did not in several different cells, we assessed PCSK9 activity in another

hepatic (HuH7) and fibroblast (917) cells (Figure 3.5).

Similarly, wild-type PCSK9 and the mutant PCSK9-D374Y was added to the medium

of cells cultured in LDLR expression-induced medium. After 6-hour incubation, cells were

collected and subjected to flow cytometry using AlexaFluor® 488-labeled C7 antibody to

detect LDLRs. C7 antibody specifically binds to the first cysteine-rich repeat of the LDLR

ligand-binding domain (111). Although permeabilization steps were not performed, we could

not completely exclude the possibility that C7 antibody might go into the cells and recognize

endocytosed LDLRs. So, LDLR levels in this experiment were considered as whole cell

proteins.

47

Figure 3.4. Increased incubation time with PCSK9 had no effects on the resistance of SV589 human fibroblast cells to PCSK9-mediated LDLR degradation. (A) HepG2 cells were cultured to ~80% confluence in medium F. The medium was then switched to sterol-depleting medium G to induce LDLR expression. Wild-type PCSK9 (5, 10, and 20 µg/ml) and PCSK9-D374Y (2 µg/ml) were added to the medium for 18 hours. Cell surface proteins were biotinylated as described in Materials and Methods. Whole cell lysates and streptavidin-precipitated proteins were subjected to 8% SDS-PAGE followed by immunoblot analysis of LDLR, PCSK9 and TFR. Secondary infrared dye (IRDye800)-labeled antibodies were used for detection on the LI-COR Odyssey infrared imaging system. (B) SV589 cells were cultured to ~80% confluence in medium B, and then were incubated overnight in sterol-depleting medium C to induce LDLR expression. SV589 cells were treated with PCSK9 and whole cell proteins as well as cell surface proteins were analyzed as described in Panel A. All experiments were performed twice with similar results.

48

Figure 3.5. PCSK9-mediated LDLR degradation assay in HuH7 human hepatoma cells and 917 human fibroblast cells. (A) HuH7 cells were cultured in medium B to ~80% confluence, and the medium were subsequently switched to lipoprotein-deficient medium C for 18 hours to induce LDLR expression. Following wild-type PCSK9 (5, 10, 20 µg/ml) and PCSK9-D374Y (2 µg/ml) incubation for 6 hours, cells were fixed as described in Materials and Methods. AlexaFluor® 488-labeled C7 antibody was used to detect cell surface LDLRs, and the cells were incubated overnight before being collected. (B) 917 human fibroblast cells were treated and samples were prepared as described in Panel A. Samples were analyzed on BD FACSAria flow cytometer to quantify the signal of LDLRs in each population. LDLR levels were corrected to background, and values were expressed relative to control cells (no addition – NA). Graphical representations are the average and standard deviation from 3 different experiments. * indicates a statistical difference between columns with significance p <0.05 by Student t-test; ** p<0.005.

49

Treatment of HuH7 cells with 5 µg/ml wild-type PCSK9 lowered LDLR levels by

~60% whereas LDLRs in cells treated with 20 µg/ml wild-type PCSK9 were 80% less than

untreated cells. Here, we were unable to observe the PCSK9 concentration-dependent

manner of LDLR decrease in response to exogenous purified PCSK9 as well as the exact 10-

fold greater activity of the mutant PCSK9-D374Y in degrading LDLRs, perhaps due to

association of C7 antibody to a portion, not whole, of internalized LDLRs in addition to cell

surface LDLRs.

However, PCSK9 failed to degrade LDLRs in fibroblast cells again. When 917

fibroblast cells were incubated with either 20 µg/ml wild-type PCSK9 or 2 µg/ml PCSK9-

D374Y, LDLR levels were not notably reduced compared to untreated cells.

3.4 PCSK9 endocytosis were LDLR-dependent in both hepatic and fibroblast cells

It was shown that PCSK9 internalization in hepatic cells required the LDLR (7, 99).

Moreover, the above results showed that the mutant PCSK9-D374Y, which was identified to

have 5 to 30-fold greater affinity in binding to the LDLR (93, 96), was taken up more

efficiently than wild-type PCSK9 in both hepatic and fibroblast cells, suggesting the

requirement of the LDLR for PCSK9 uptake in these cell types. To assess the role of the

LDLR in PCSK9 internalization, we measured AlexaFluor® 488-labeled PCSK9 uptake in

the absence and presence of the LDLR.

For this and subsequent experiments, HepG2 cells were used as a representative model

for hepatic cells whereas SV589, which are referred to fibroblast cells from now, represented

fibroblasts. These cell types were incubated either in medium containing statin (pravastatin)

to induce LDLR expression or in medium containing sterols (cholesterol and 25-

hydroxycholesterol) to suppress LDLR expression for 18 hours. AlexaFluor® 488- labeled

50

PCSK9-D374Y (1 µg/ml) was allowed to be taken up for 1 hour in the continuous presence

of E64 before the cells were washed with stripping buffer to remove cell surface labeled

PCSK9. E64 is a potent and highly selective cysteine protease inhibitor that was shown to

completely abolish PCSK9-mediated LDLR degradation in lysosomes (102). Samples were

then subjected to flow cytometry to measure the overall population of cells that had positive

signal for internalized labeled PCSK9.

After 1-hour incubation, AlexaFluor® 488-labeled PCSK9-D374Y was detectable in

approximately 70% of fibroblast cells expressing LDLRs while the amount of internalized

PCSK9-D374Y was dramatically reduced in fibroblast cells lacking LDLR expression

(Figure 3.6). Similarly, abundant labeled PCSK9 was found to associate with approximately

50% of hepatic cells expressing LDLRs, and PCSK9 endocytosis was significantly abolished

in these cells when LDLR expression was suppressed. However, a small amount of

AlexaFluor® 488-labeled PCSK9-D374Y was still detectable in either hepatic or fibroblast

cells deficient in LDLRs, perhaps due to incomplete sterol-dependent suppression of LDLR

expression. Also, it was possible that another receptor might play only a minor role while

internalization of exogenous PCSK9 in hepatic and fibroblast cells was mostly dependent on

the LDLR.

To further confirm the importance of LDLR in PCSK9 endocytosis, we next tested

PCSK9 association/uptake into cells expressing either wild-type LDLR or a PCSK9 binding-

defective LDLR (LDLR-E296Q). This mutation, which would presumably decrease calcium-

affinity of the LDLR EGF-A domain more extensively according to the equivalent residue in

coagulation factor IX (112), failed to bind PCSK9 at neutral pH as well as at acidic

51

Figure 3.6. PCSK9 uptake in the presence and absence of LDLRs in HepG2 human hepatoma cells and SV589 human fibroblast cells. SV589 cells were grown in medium B to ~80% confluence, and then were cultured overnight in sterol-depleting medium C or medium D containing sterols (25-hydroxycholesterol/cholesterol) to up-regulate and down-regulate LDLR expression, respectively. Similarly, HepG2 cells were cultured in sterol-depleting medium G or medium H containing sterols (25-hydroxycholesterol/cholesterol) for 18 hours. These mediums were supplemented with 150 µM E64 to inhibit lysosomal degradation. AlexaFluor® 488-labeled PCSK9-D374Y was added to the medium at 1 µg/ml for 1 hour before the medium was replaced with stripping buffer to wash cell surface PCSK9. Cells were collected and subjected to analysis using the BD FACSAria flow cytometer. The signal of internalized PCSK9 in each population was quantified and corrected to background. Graphical representations are the average and standard deviation from 3 different experiments. ** indicates a statistical difference between columns with significance p <0.005 by Student t-test.

52

pH in in vitro binding studies (data not shown) because the interaction between PCSK9 and

the LDLR EGF-A domain is calcium-dependent as stated in the introduction (91). LDLR-

transfected cells were treated overnight with sterols (25-hydroxycholesterol and cholesterol)

to suppress endogenous LDLR expression prior to incubation with 1 µg/ml purified PCSK9-

D374Y for 6 hours. E64 was used to inhibit lysosomal degradation of internalized PCSK9.

PCSK9 association and uptake occurred normally in cells expressing wild-type LDLR,

but was completely negligible in cells expressing the mutant LDLR-E296Q although both

the wild-type LDLR (lane 7-8) and the mutant LDLR-E296Q (lane 5-6) were expressed

normally on the cell surface (Figure 3.7). Purified added PCSK9 was detected in whole cell

extracts (lane 4), but not in cell surface proteins (lane 8) of cells expressing wild-type LDLR,

suggesting that most of cell-associated PCSK9 was internalized as usual. Particularly, not

only hepatic cells but also fibroblast cells expressing wild-type LDLR could normally

associate and then uptake exogenous purified PCSK9 after a 6-hour incubation (lane 4).

Nevertheless, no traces of PCSK9 were detected in either whole cell lysates (lane 2) or

membrane proteins (lane 6) of hepatic and fibroblast cells expressing the binding-defective

mutation (LDLR-E296Q), indicating that PCSK9 failed to associate as well as be taken up

into these cells in the absence of LDLR/PCSK9 interaction. Combined together, these data

strongly confirmed the LDLR-dependent manner of PCSK9 endocytosis in both hepatic and

fibroblast cells.

3.5 Both wild-type PCSK9 and the mutant PCSK9-D374Y trafficked to lysosomes in

hepatic cells

To elucidate the mechanisms responsible for different responses to PCSK9-mediated

LDLR degradation in hepatic and fibroblast cells, first we wanted to determine the fate of

53

Figure 3.7. PCSK9 uptake in cells expressing either wild-type LDLR or a PCSK9 binding-defective mutation. (A) HepG2 cells were grown in medium F to ~60-70% confluence. The cells were then transiently transfected with wild-type LDLR or LDLR-E296Q expression vectors, and incubated overnight in sterol-supplemented medium (25-hydroxycholesterol/cholesterol) to suppress endogenous LDLR expression. PCSK9-D374Y (1 µg/ml) was incubated for 6 hours in the continuous presence of 150 µM E64. Cell surface proteins were isolated by biotinylation as described in Materials and Methods. Whole cell lysates and membrane proteins were subjected to immunoblot analysis of LDLR, PCSK9 and TFR. Secondary infrared dye (IRDye800)-labeled antibodies were used for detection on the LI-COR Odyssey infrared imaging system. (B) SV589 cells were cultured to ~60% confluence in medium B. The cells were treated and whole cell proteins as well as cell surface proteins were analyzed as described in Panel A. All experiments were performed twice with similar results.

54

PCSK9 after binding to the LDLR in these cells.

PCSK9 internalization, which depended on its interaction with the LDLR on the cell

surface, led to a dramatic reduction of LDLRs in hepatic cells through routing the receptor to

lysosomes for degradation (7, 99, 102), possibly along with PCSK9. PCSK9 trafficking was

characterized using 125I-labeled PCSK9 degradation assay along with co-localization studies.

Hepatic cells were cultured in sterol-deficient medium supplemented with statin (pravastatin)

for 18 hours prior to radioisotope-labeled PCSK9 incubation. We monitored the fate of both

wild-type PCSK9 and the mutant PCSK9-D374Y, which was used at 5-fold lesser

concentration compared to the wild-type. After 1-hour incubation, residual cell surface

PCSK9 was removed, and fresh medium was added. Following 6-hour chase of internalized

125I-labeled PCSK9, the amount of mono-iodotyrosine radioactivity (TCA-soluble) in the

medium was measured as a catabolic product of 125I-labeled PCSK9 degradation in

lysosomes. C7 monoclonal antibody, which is taken up by the LDLR, and subsequently,

dissociates from the receptor in early endosomes and proceeds for degradation in lysosomes,

was used as a positive control for lysosomal degradation (111).

After 6 hours, TCA-soluble counts were significantly increased in the medium for both

wild-type PCSK9 and the mutant PCSK9-D374Y (Figure 3.8). The levels of 125I-mono-

iodotyrosine released into the medium reach ~4,000 cpm/mg protein for wild-type PCSK9,

similar to those of the mutant PCSK9-D374 (~3,800 cpm/mg protein). Of note, labeled

PCSK9-D374Y was used at concentrations 5-fold less than those of wild-type PCSK9.

Degradation of 125I-labeled C7 antibody attained ~15,000 cpm/mg protein after 6 hours,

confirming lysosomal degradation was effective under these conditions. These data indicated

that in hepatic cells, wild-type PCSK9 and the mutant PCSK9-D374Y were internalized and

delivered to lysosomes for degradation, in which the mutant was degraded more effectively.

55

Figure 3.8. 125I-labeled PCSK9 degradation assay. HepG2 cells were cultured to ~80% confluence in medium F. The medium was switched to sterol-depleting medium G, and the cells were incubated for 18 hours to induce LDLR expression. Similarly, SV589 cells were cultured in sterol-depleting medium C. Medium A containing ~5 µg/ml 125I-labeled wild-type PCSK9 or ~1 µg/ml 125I-labeled PCSK9-D374Y was then added to the cells for 1 hour. In negative controls, the cells were treated with 100 µM chloroquine 30 minutes prior to and during later incubation to inhibit lysosomal degradation. The medium was removed, and the cells were incubated in label-free medium A for 6 hours after being washed extensively. The amounts of 125I-mono-iodotyrosine were determined in the medium as described in Materials and Methods. TCA-soluble counts were normalized to protein levels, and corrected to negative controls. Graphs represent the means ± standard deviation from three independent experiments.

56

Co-localization studies between PCSK9 and lysosomal markers also demonstrated that

in hepatic cells, both wild-type PCSK9 and the mutant D374Y trafficked to lysosomes after

being taken up into these cells via the LDLR (Figure 3.9). In this experiment, following

incubation in sterol-depleting medium containing statin (pravastatin), cells were treated with

AlexaFluor® 488-labeled PCSK9 for 1 hour and then washed extensively. Labeled PCSK9

were chased for 6 hours in the continuous presence of E64 while LysoTracker, the lysosome

marker, was added 2 hours before being directly observed on the confocal microscope. E64

served as a lysosomal protease inhibitor to inhibit degradation of internalized labeled PCSK9

(102). Addition of AlexaFluor® 488-labeled PCSK9 to the medium supplemented with E64

resulted in the diffuse localization of these proteins in big punctate perinuclear structures of

hepatic cells. Notably, a significant amount of these structures overlapped with LysoTracker

signal after 6 hours for both labeled wild-type PCSK9 and the mutant PCSK9-D374Y,

suggesting that these proteins proceeded to lysosomes for degradation after binding to the

LDLR.

3.6 Wild-type PCSK9, not the mutant PCSK9-D374Y, was completely degraded in

fibroblast cells

Above results showed that PCSK9 failed to degrade LDLRs in fibroblast cells despite

its normal LDLR-dependent cellular uptake; however, the fate of PCSK9 after binding to the

LDLR had been still unknown. To explore whether PCSK9 was degraded in fibroblast cells

as it is in hepatic cells whereas the LDLR recycled to the cell surface, we also performed

125I-labeled PCSK9 degradation assay.

Fibroblast cells were cultured as described above. Figure 3.8 showed that following 6-

hour chase of internalized 125I-labeled wild-type PCSK9, the levels of TCA-soluble proteins

57

Figure 3.9. Co-localization of PCSK9 and the lysosome marker (LysoTracker) in HepG2 human hepatoma cells. HepG2 cells were seed in µ-slide 8-well plates to ~80% confluence, and then were cultured overnight in lipoprotein-deficient medium G supplemented with 150 µM E-64. The medium was replaced with medium containing 5 µg/ml AlexaFluor® 488-labeled PCSK9-D374Y, and the cells were incubated for 1 hour. Internalized AlexaFluor® 488-labeled PCSK9 was then chased for 6 hours in label-free medium G containing 150 µM E-64 to inhibit lysosomal degradation. LysoTracker® Red DND-99 was diluted to the final working concentration (200 nM), and incubated for 2 hours prior to two washes with label-free medium E and direct observation on microscope at the indicated time. Images were taken using the confocal microscope Fluoview FV1000 Version 2.1 (Olympus), in which PCSK9 was visualized using the 488nm laser, and the 543nm laser was used for LysoTracker.

58

released in the medium reached ~4,500 cpm/mg protein, equivalent to those of hepatic cells.

Therefore, wild-type PCSK9 was internalized as well as degraded equally in both hepatic

and fibroblast cells, indicating dissociation of wild-type PCSK9 from the recycling LDLR in

fibroblast cells. Nevertheless, only trace amount of 125I- mono-iodotyrosine (<500 cpm/mg

protein) was detected in the medium of fibroblast cells treated with radioisotope-labeled

PCSK9-D374Y. Particularly, this amount was considerably lower than the amount of 125I-

mono-iodotyrosine observed in hepatic cells treated with labeled PCSK9-D374Y, suggesting

that high percentage of internalized PCSK9-D374Y was degraded in hepatic cells, but not in

fibroblast cells. Moreover, C7 antibody was degraded to the same extent (~15,000-16,000

cpm/mg protein) in both of hepatic and fibroblast cells, verifying that lysosomal degradation

functioned properly in these cells. Combined together, these data suggested that different

from hepatic cells, wild-type PCSK9 separated from the recycling LDLR and went to

lysosomes for degradation in fibroblast cells whereas the mutant PCSK9-D374Y seemed to

maintain the binding to the receptor and recycle to the cell surface. Another possibility is that

similar to wild-type PCSK9, the mutant also dissociated from the recycling LDLR in

fibroblast cells, but still stuck in endocytic compartments instead of proceeding to lysosomes

for degradation.

3.7 More internalized PCSK9-D374Y proteins recycled to the cell surface

3.7.1 Recycling assay

For further determination of PCSK9-D374Y fate in fibroblast cells, we turned to a

recycling assay based on on-cell western to quantify the percentage of recycled and

intracellular PCSK9 compared to total internalized proteins (Figure 3.10).

59

Figure 3.10. Recycling assay of wild-type PCSK9 and PCSK9-D374Y in SV589 human fibroblast cells. (A) SV589 cells were cultured in 12-well plates to ~80% confluence, and then were incubated in serum-free medium A for >1 hour to deplete endogenous transferrin. For monensin-treated wells, the cells were also treated with 50 µM monensin for >1 hour prior to and during later incubation to inhibit the recycling process. Biotin-labeled transferrin was incubated with IRDye® 800CW Streptavidin in medium A for 1 hour at 37oC, and the complexes were added to the cells for 1 hour. Subsequently, the cells were washed at 4oC with 20 mM TCEP to remove cell surface labeled complexes before incubation in medium A supplemented with 20 mM TCEP for 2 hours. The plate was directly scanned on the LI-COR Odyssey infrared imaging system at the indicated time intervals. (B) The recycling assay was performed as described above, with the exception that the internalized complexes were chased for 6 hours in the medium continuously containing TCEP and E-64. Signal intensity was quantified using Odyssey 2.0 software. PCSK9 levels were normalized to DNA levels stained by DRAQ5TM (1:10,000), and corrected for background using untreated wells. Graphs represent the means ± standard deviation from three independent experiments.

60

After incubation in lipoprotein-deficient medium supplemented with statin to induce

LDLR expression, cells were allowed to uptake IRDye® 800CW-Streptavidin/Biotin-

PCSK9 complexes for 1 hour, and then washed extensively at 4oC to remove remaining

complexes from the cell surface. Internalized PCSK9 was directly visible in fibroblast cells

due to the IRDye® 800CW tag of streptavidin as well as the interaction between streptavidin

and biotin particles attached to PCSK9. The signal of internalized PCSK9 was chased in the

medium continuously containing E64 and TCEP, and was measured at various intervals over

the ensuing 6 hours. E64 was used to inhibit lysosomal degradation whereas TCEP served as

a reducing agent that disrupted the disulfide bond between PCSK9 and biotin particles,

leading to cleavage of the IRDye® 800CW-Streptavidin-Biotin tag from PCSK9. Therefore,

if PCSK9 recycled to the cell surface, we would see the incremental loss of signal over time.

In contrast, if PCSK9 still stayed in the cells, we would see no changes in the signal of

internalized PCSK9.

Transferrin, a well-known recycling protein, was used as a positive control to verify

the preciseness of this experiment. Exogenous diferric transferrin specifically binds the

transferrin receptor on the cell surface, and the complexes are subsequently internalized via

the receptor-mediated endocytosis. At the reduced pH in early endosomes, iron dissociates

from transferrin and still stays within the cells while transferrin remains bound to the

transferrin receptor and is recycled to the cell surface (113). Consistent with this scenario,

our recycling assay showed that during the chase period in the continuous presence of TCEP,

the signal of internalized transferrin dropped gradually in fibroblast cells (Figure 3.10).

Particularly, almost the entire signal was lost after 120 minutes, indicating recycling of

transferrin to the cell surface. Moreover, this incremental decrease in signal was completely

61

abolished when fibroblast cells were treated with 50 µm monensin, a recycling inhibitor

(114).

For wild-type PCSK9, no significant changes were observed in the intensity of

internalized proteins after a 6-hour chase in the medium continuously supplemented with

TCEP, suggesting that higher percentage of internalized wild-type PCSK9 remained inside

fibroblast cells, instead of recycling to the cell surface. Here, although IRDye® 800CW was

not usually degraded, but accumulated in the cells, we still used E64 on the safe side to

inhibit lysosomal degradation of internalized PCSK9. This was consistent with the results of

125I-labeled PCSK9 degradation assay, which indicated that wild-type PCSK9 separated from

the recycling LDLR and trafficked to lysosomes for degradation. Recycling assay showed

that only <10% of wild-type PCSK9 signal was lost during 6 hours, which might represent

the release of residual labeled complexes from the cell surface into the medium. In contrast,

>60% of PCSK9-D374Y signal was lost following 6-hour chase, suggesting that higher

percentage of internalized PCSK9-D374Y still bound the LDLR and recycled to the cell

surface along with the receptor.

3.7.2 Co-localization studies

Co-localization studies that showed both wild-type PCSK9 and mutant PCSK9-D374Y

co-localized with LysoTracker in fibroblast cells further confirmed the above results.

However, PCSK9-D374Y also overlapped with transferrin in recycling compartments while

wild-type PCSK9 did not.

In imaging studies between AlexaFluor® 488-labeled PCSK9 and LysoTracker,

fibroblast cells were cultured as described above, with the exception that the percentage of

overlapped pixels was quantified at intervals during a 6-hour chase in the medium

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supplemented with E64. In fibroblast cells, wild-type PCSK9 (Figure 3.11) and the mutant

PCSK9-D374Y (Figure 3.12) overlapped with LysoTracker in large punctate vacuoles, and

PCSK9 co-localization with LysoTracker increased time-dependently. Especially, 2-hour

chase resulted in ~50% of intracellular PCSK9 that overlapped LysoTracker, whereas the

percentage of co-localization between PCSK9 and LysoTracker reached >80% after 6-hour

chase. Although the above results showed that more than half of PCSK9-D374Y recycled to

the cell surface in fibroblast cells, the percentage of co-localization between PCSK9-D374Y

and LysoTracker over time was roughly equal to that of wild-type PCSK9. One explanation

is that recycled PCSK9-D374Y could release into the medium and be eventually removed

whereas the percentage of co-localization with LysoTracker was quantified based on the

intracellular signal of labeled PCSK9. The majority of PCSK9-D374Y signal overlapped

with LysoTracker was intracellular PCSK9 that did not recycle to the cell surface.

For labeling recycling compartments, we used AlexaFluor® 647-labeled transferrin.

Cells were pulsed with labeled transferrin and PCSK9 for 1 hour following incubation in

serum-free medium to deplete endogenous transferrin. Labeled proteins were subsequently

chased in the medium supplemented with E64 to block lysosomal degradation of internalized

PCSK9. After 2-hour chase, AlexaFluor® 647-labeled transferrin concentrated in clusters of

small vacuoles, which significantly overlapped with the signal of labeled PCSK9-D374Y

(Figure 3.13). No significant co-localization between wild-type PCSK9 and transferrin was

observed in fibroblast cells under similar conditions. Furthermore, we also used Rab4, a

well-known early endosome marker, as a control to indicate that AlexaFluor® 647-labeled

transferrin was chased out of early endosomes after 2-hour chase, and that the co-localization

63

Figure 3.11. Co-localization of wild-type PCSK9 and the lysosome marker (LysoTracker) in SV589 human fibroblast cells. SV589 cells were cultured in µ-slide 8-well plates to ~80% confluence, and then were incubated overnight in sterol-depleting medium C supplemented with 150 µM E-64. 30 µg/ml AlexaFluor® 488-labeled wild-type PCSK9 was added to the medium for 1 hour. Internalized labeled PCSK9 was then chased for 6 hours in label-free medium C containing 150 µM E-64. LysoTracker® Red DND-99 was diluted to the final working concentration (200 nM), and incubated for 2 hours prior to direct observation on microscope at the indicated time. Images were taken on the confocal microscope Fluoview FV1000 Version 2.1 (Olympus), in which PCSK9 was visualized using the 488nm laser and the 543nm laser was used for LysoTracker. The percentage of co-localization was quantified using Image J (http://rsb.info.nih.gov/ij/), and corrected for background using negative images. A minimum of 3 fields was analyzed per indicated time. Graphs represent the means ± standard deviation from three independent experiments.

64

Figure 3.12. Co-localization of PCSK9-D374Y and the lysosome marker (LysoTracker) in SV589 human fibroblast cells. SV589 cells were cultured in µ-slide 8-well plates to ~80% confluence, and were incubated overnight in sterol-depleting medium C supplemented with 150 µM E-64. 5 µg/ml AlexaFluor® 488-labeled PCSK9-D374Y was added to the medium for 1 hour. Internalized labeled PCSK9 was then chased for 6 hours in label-free medium C containing 150 µM E-64. LysoTracker® Red DND-99 was diluted to the final working concentration (200 nM) and incubated for 2 hours prior to direct observation on microscope at the indicated time. Images were taken on the confocal microscope Fluoview FV1000 Version 2.1 (Olympus), in which PCSK9 was visualized using the 488nm laser and the 543nm laser was used for LysoTracker. The percentage of co-localization was quantified using Image J (http://rsb.info.nih.gov/ij/), and corrected for background using negative images. A minimum of 3 fields was analyzed per indicated time. Graphs represent the means ± standard deviation from three independent experiments.

65

Figure 3.13. Co-localization of the mutant PCSK9-D374Y and transferrin in recycling compartments of SV589 human fibroblast cells. (A) SV589 cells were grown in µ-slide 8-well plates to ~80% confluence, and were cultured overnight in medium C supplemented with 150 µM E-64. Following >1 hour incubation in serum-free medium A to deplete endogenous transferrin, cells were treated with AlexaFluor® 488-labeled PCSK9-D374Y (or AlexaFluor® 488-labeled wild-type PCSK9) and AlexaFluor® 647-labeled transferrin for 1 hour. The labeled proteins were then chased for 2 hour in label-free medium A. (B) SV589 cells were transiently transfected with tRFP tagged Rab4 expression plasmid using Lipofectamine 2000. After 20 to 24 hours, the cells were incubated in serum-free medium A for >1 hour, followed by the addition of AlexaFluor® 647-labeled transferrin for 1 hour. The cells were then chased for 2 hours in label-free medium A. Images were directly taken on the confocal microscope Fluoview FV1000 Version 2.1 (Olympus) using the 488nm laser for PCSK9, the 543nm laser for Rab4, and the 633nm laser for transferrin. The percentage of co-localization was quantified using Image J (http://rsb.info.nih.gov/ij/), and corrected for background using negative images. Graphs represent the means ± standard deviation from three independent experiments.

66

between the mutant PCSK9-D374Y and transferrin really happened in recycling

compartments, but not in early endosomes.

3.8 LDLR-EGF66 variant that binds PCSK9 in a calcium-independent manner could

restore wild-type PCSK9 activity in fibroblast cells

PCSK9 failed to promote LDLR degradation in fibroblast cells despite its normal

uptake into these cells. Above results showed that in fibroblast cells, wild-type PCSK9

dissociated from the recycling receptor and proceeded to lysosomes for degradation whereas

the mutant PCSK9-D374Y, which has higher affinity in binding to the LDLR, seemed to

maintain the binding to the LDLR in early endosomes and recycle to the cell surface along

with the receptor. However, the mechanisms responsible for different PCSK9 fate in hepatic

and fibroblast cells had not been molecularly defined.

As discussed in the introduction, PCSK9 binds to the LDLR in a calcium-dependent

manner (91). Here, we examined PCSK9 activity in fibroblast cells transiently

overexpressing either wild-type LDLR or a special LDLR variant (LDLR-EGF66) that binds

PCSK9 in a calcium-independent manner. This variant contains five single-site mutations in

the EGF-A domain of the LDLR, including Aspartate-299-Alanine, Asparagine-301-

Leucine, Valine-307-Isoleucine, Asparagine-309-Arginine, and Aspartate-310-Lysine. All of

them are involved in PCSK9 binding, and therefore isolated EGF66 exhibits >10-fold

stronger binding affinity toward PCSK9 compared to the wild type EGF-A domain.

Especially, it should be noted that the interaction of LDLR-EGF66 with PCSK9 would no

longer be dependent on calcium (115). LDLR-transfected cells were treated with sterols (25-

hydroxycholesterol and cholesterol) to suppress endogenous LDLR expression prior to 6-

hour incubation with wild-type PCSK9 (10 µg/ml) or PCSK9-D374Y (2 µg/ml). Non-

67

transfected cells were used as controls for the resistance of fibroblast cells to PCSK9-

mediated LDLR degradation.

As shown in Figure 3.14, fibroblast cells expressing either wild-type LDLR or the

LDLR-EGF66 variant were still highly resistant to LDLR degradation promoted by PCSK9-

D374Y, whereas LDLR(EGF66)-transfected cells became partially responsive to wild-type

PCSK9. Following PCSK9-D374Y incubation, LDLR protein levels were not significantly

decreased (only ~10% compared to untreated cells) in fibroblast cells expressing wild-type

LDLR (lane 5) or LDLR-EGF66 (lane 8). LDLR levels in non-transfected cells were

approximately 20% less than those of untreated cells as usual (lane 2). Similarly, no

reduction in cellular LDLRs was detected in non-transfected cells (lane 3) as well as cells

expressing wild-type LDLR (lane 6) after treatment with wild-type PCSK9. However,

approximately half of LDLR levels were decreased in cells expressing the LDLR-EGF66

variant (lane 9) when these cells were incubated with 10 µg/ml wild-type PCSK9. Both wild-

type PCSK9 and the mutant PCSK9-D374Y occurred at similar levels in cells expressing

wild-type LDLR or LDLR-EGF66, indicating that PCSK9 binding and uptake was mediated

equally by either construct.

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Figure 3.14. Wild-type LDLR and LDLR-EGF66 degradation mediated by PCSK9 in SV589 human fibroblast cells. SV589 cells were grown in medium B to ~60-70% confluence. The cells were then transiently transfected with wild-type LDLR or LDLR-EGF66 expression vectors, and incubated overnight in sterol-supplemented medium (25-hydroxycholestrol/cholesterol) to suppress endogenous LDLR expression. Wild-type PCSK9 (10 µg/ml) and PCSK9-D374Y (2 µg/ml) was incubated for 6 hours. Whole cell lysates were subjected to 8% SDS-PAGE followed by immunoblot analysis of LDLR, PCSK9 and TFR. Secondary detection used infrared dye (IRDye800)-labeled antibodies. Blots were visualized and quantified using the LI-COR Odyssey infrared imaging system. LDLR levels were normalized to TFR expression and expressed relative to control cells (no addition – NA). Graphs represent the means ± standard deviation from three independent experiments. * indicates a statistical difference between columns with significance p <0.05 by Student’s t-test; ** p<0.005.

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4. Discussion

Increased plasma levels of LDL-C represent the greatest risk factor of CHD (1). The

LDLR that mediates the endocytic uptake of LDL particles into cells is the major

determinant of plasma LDL-C concentrations (42, 58). The discovery of PCSK9, a secreted

protein that promotes LDLR degradation in liver, has opened a new era not only for our

understanding of cholesterol metabolism but also for the treatment of cardiovascular

diseases. Particularly, the strikingly low levels of LDL-C found in individuals who carry

loss-of-function mutations in PCSK9 have inspired researchers to investigate the

mechanisms responsible for PCSK9-mediated LDLR degradation (8). In the present study,

we determined PCSK9 activity on LDLR levels of several cell lines, including hepatic cells

and fibroblast cells. Our hypothesis is that PCSK9 has cell-type specificity, in which hepatic

cells are the most responsive cells. We also illuminated the fate of PCSK9 after binding to

the LDLR in nonresponsive cells, which might allow us to explore potential mechanisms that

lead to dissimilar responses to PCSK9-mediated LDLR degradation in hepatic and fibroblast

cells. These mechanisms could be targeted to inhibit PCSK9 activity and preserve LDLR

function in the liver.

4.1 PCSK9 degrades LDLRs in a cell-type dependent manner

It was shown that injection of human recombinant PCSK9 into mice and adenovirus-

mediated overexpression of PCSK9 in mice resulted in a dramatic reduction of LDLRs in

liver as well as LDLRs in kidney, ileum and lung, in a lesser extent. However, no changes in

LDLR levels were observed in brains (67, 71, 116, 117). Particularly, even when PCSK9 was

specifically overexpressed in kidney, LDLR levels in this tissue were still reduced to a lower

degree than those in liver (118). In addition, purified PCSK9 introduced into the circulation

of wild-type mice at physiological concentrations induced robust degradation of hepatic

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LDLRs whereas it was completely ineffective to promote LDLR degradation in the adrenal

glands (106), which also have the highest LDLR expression in the body for cholesterol

uptake and are the main places to produce steroid hormones in humans (119). These

findings, when taken together, suggested that not all tissues respond equally to circulating

PCSK9, in which liver is most responsive. Here we showed that incubation of liver-derived

cell-lines with purified PCSK9 caused dramatic degradation of cell surface as well as whole

cell LDLRs (Figure 3.3 and 3.5), whereas PCSK9 only had minimal effects on LDLRs in

several lines of fibroblast cells (Figure 3.2 and 3.5). Thus, PCSK9 degrades LDLRs in a cell-

type specific manner. Our data are consistent with those reported that recombinant human

PCSK9 added to the medium did not significantly affect the protein levels of LDLR in

mouse embryonic fibroblasts whereas it promoted LDLR degradation in hepatocytes (7).

Particularly, addition of purified PCSK9 to the medium at levels comparable with those in

human plasma induced LDLR degradation in hepatic cells, but not in fibroblast cells (Figure

3.1), confirming that cell-type specificity of PCSK9 does occur physiologically. Besides,

SV589 cells were still highly resistant to PCSK9-mediated LDLR degradation after

overnight incubation with exogenous PCSK9 (Figure 3.4). Therefore, it is not likely that

dissimilar responses to PCSK9 in hepatic and fibroblast cells mainly result from different

association and uptake time of PCSK9 in these cells.

However, it was shown that although PCSK9 was not involved in the degradation of

LDLRs in adult mouse brain, PCSK9 seemed to affect LDLR levels in brain during

development and following transient ischemic stroke (120). Therefore, the effect of PCSK9

on LDLRs might be not the same under different conditions. Studies using more cell lines

under a wide range of conditions will help to further address this hypothesis.

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4.2 PCSK9 association/uptake is LDLR-dependent in both of hepatic and fibroblast

cells

Purified PCSK9 injected into wild-type mice had a half-life of 5 minutes whereas this

interval was increased to 15 minutes in LDLR-/- mice, suggesting that functional LDLRs

might play an important role in mediating PCSK9 clearance (106). Complementing this

experiment, numerous studies have demonstrated that PCSK9 internalization in hepatic cells

is mediated by the LDLR. LDLR deficiency in hepatocytes from LDLR-null mice as well as

RNA interference-mediated knockdown of LDLR significantly inhibited PCSK9 endocytosis

(99, 101). As indicated in LDLR degradation assay, PCSK9 failed to degrade LDLRs in

fibroblast cells despite its normal uptake into the cells (Figure 3.1, 3.2, and 3.4). However, it

could be argued that PCSK9 was taken up via different receptors, which led to the lack of

PCSK9-mediated LDLR degradation in fibroblast cells. To rule out this possibility, we

examined the contribution of LDLR to PCSK9 association/uptake in hepatic as well as

fibroblast cells. A high percentage of exogenous labeled PCSK9 was internalized in hepatic

and fibroblast cells expressing LDLRs whereas the amount of endocytosed PCSK9 was

completely abolished when LDLR expression was suppressed in these cells (Figure 3.6).

Moreover, no cell-associated PCSK9 along with internalized PCSK9 were detected in either

hepatic or fibroblast cells expressing a binding-defective LDLR (LDLR-E296Q) (Figure

3.7). This mutation, which presumably decreased calcium-affinity of the LDLR EGF-A

domain more extensively, failed to bind PCSK9 (data not shown) because of the calcium-

dependent manner of PCSK9 binding to the LDLR (91, 112). Combined together, these data

confirmed that PCSK9 association/uptake is LDLR-dependent in hepatic and fibroblast cells.

Similar to our results, it was shown that purified added PCSK9 was taken up by mouse

embryonic fibroblasts in a manner that depended on the LDLR (7). Besides, PCSK9 failed to

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internalize into cells expressing a mutant LDLR missing the EGF-A domain, which is

necessarily required for the interaction between PCSK9 and the LDLR (91). However, these

data, when taken together, argued against a recent study that reported PCSK9 was

endocytosed in an LDLR-independent manner in HepG2 cells (121).

4.3 PCSK9 in hepatic cells

To explore the potential mechanisms underlying dissimilar responses to PCSK9-

mediated LDLR degradation in hepatic and fibroblast cells, we examined the fate of PCSK9

after binding to the LDLR in these cell lines.

Previous work demonstrated that after being secreted from liver as a stable complex

with its prosegment (72), PCSK9 binds to the LDLR on the cell surface (7, 101). The

LDLR/PCSK9 complex is then internalized via the same endocytic machinery importing

LDL, which requires the endocytic adaptor protein ARH (7, 99). However, different from

bound lipoprotein particles that release from the LDLR in the low pH and low calcium

environment in early endosomes (42, 58, 65), PCSK9 still binds to the LDLR with

considerably increased affinity (92), prevents the receptor from recycling to the cell surface,

and directs it to lysosomes where the whole complex, including the LDLR and PCSK9, is

degraded (7, 102, 104) (Figure 1.4).

Results obtained in our studies confirmed this scenario in hepatic cells. PCSK9

degradation assay revealed that the levels of mono-iodotyrosine radioactivity were

significantly increased in the medium following 6-hour chase of internalized 125I-labeled

wild-type PCSK9 and PCSK9-D374Y, implicating that both of wild-type PCSK9 and the

mutation were delivered to lysosomes for degradation in hepatic cells. Particularly, PCSK9-

D374Y was degraded more effectively than wild-type PCSK9 (about 5-fold) as indicated by

the different concentrations of labeled proteins added to the medium and the nearly equal

73

amounts of TCA-soluble counts released in the medium (Figure 3.8). In addition, wild-type

PCSK9 and the mutant PCSK9-D374Y co-localized with the lysosome marker

(LysoTracker) in live cell imaging, confirming the trafficking of PCSK9 to lysosomes in

hepatic cells (Figure 3.9). Therefore, in hepatic or responsive cells, both of wild-type PCSK9

and PCSK9-D374Y were internalized and delivered to lysosomes for degradations.

4.4 PCSK9 in fibroblast cells

However, the fate of PCSK9 is not the same for wild-type PCSK9 and the mutant

PCSK9-D374Y in fibroblast cells. A significant amount of 125I-labeled wild-type PCSK9

was delivered to lysosomes for degradation in fibroblast cells, following the chase period in

PCSK9 degradation assay, whereas 125I-labeled PCSK9-D374Y was degraded at lower levels

in fibroblast. Particularly, wild-type PCSK9 was internalized and degraded equally in both of

hepatic and fibroblast cells (Figure 3.8), thus suggesting that wild-type PCSK9 separated

from the recycling receptor after being internalized in fibroblasts since the LDLR was not

readily degraded whereas PCSK9 was. Furthermore, almost no wild-type PCSK9 that

recycled to the cell surface was detected in PCSK9 recycling assay, confirming dissociation

of wild-type PCSK9 from the LDLR in fibroblast cells. However, consistent with the results

of PCSK9 degradation assay, recycling assay showed that higher percentage of internalized

PCSK9-D374Y recycled to the cell surface along with the LDLR, instead of directing the

receptor to lysosomes for degradation (Figure 3.10). Live cell imaging confirmed that both of

wild-type PCSK9 and PCSK9-D374Y co-localized with the lysosome marker (LysoTracker)

in fibroblast cells (Figure 3.11 and 3.12) while only the PCSK9-D374Y mutation also

overlapped with labeled transferrin in endocytic recycling compartments (Figure 3.13).

These data implied that even with persistent binding, PCSK9 might have reduced activity to

route the LDLR to lysosomal degradation in fibroblast cells. Similarly, it was shown that a

74

mutant PCSK9 lacking the C-terminal domain also failed to promote LDLR degradation in

hepatic cells although it still maintained the binding to the LDLR (104). The deficiency of

this mutation in degrading LDLRs has not been fully characterized. In fact, the C-terminal

domain of PCSK9 has been reported to interact with endocytic adaptor protein(s) that are

required for lysosomal targeting and PCSK9-mediated LDLR degradation (94, 105). If so,

then perhaps these same proteins or processes might be less expressed in fibroblast cells

compared to in hepatocytes. Therefore, it would be interesting to determine if C-terminal

domain truncated PCSK9 recycles in hepatocytes, similar to PCSK9-D374Y recycling in

fibroblasts.

Based on these results, our studies provide the first description of PCSK9 trafficking in

non-responsive cells, which may allow us to illuminate potential factors as well as

mechanisms that help these cells be resistant to PCSK9-mediated LDLR degradation. The

fate of PCSK9 in fibroblast cells can be described as follow. After the LDLR/PCSK9

complex is endocytosed in an LDLR-dependent manner, the LDLR does not traffic to

lysosomes for degradation, but recycles to the cell surface instead. In the case of wild-type

PCSK9, PCSK9 dissociates from the LDLR in early endosomes following internalization,

and proceeds to late endosomes/lysosomes for degradation while the receptor recycles to the

cell surface, resulting in no significant changes of LDLR levels in response to wild-type

PCSK9 (Figure 4.1). In contrast, the mutant PCSK9-D374Y has 5 to 30-fold greater affinity

in binding to the LDLR compared with wild-type PCSK9 (93, 96), so higher percentage of

PCSK9-D374Y still maintains the binding to the receptor in early endosomes. The

LDLR/PCSK9 complex then either goes to lysosomes for degradation or recycles to the cell

surface (Figure 4.2). It accounted for a small reduction of LDLRs (approximately <20%) in

response to the mutant PCSK9-D374Y.

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Figure 4.1. Model of wild-type PCSK9 in fibroblast cells. Wild-type PCSK9 directly binds the LDLR on the cell surface, and is internalized in an LDLR-dependent manner. However, different from PCSK9 fate in hepatic cells, wild-type PCSK9 dissociates from the LDLR in early endosomes, and proceeds to late endosomes/lysosomes for degradation while the receptor recycles to the cell surface. Images modified from Horton, Cohen, and Hobbs (2009)(72).

76

Figure 4.2. Model of PCSK9-D374Y in fibroblast cells. PCSK9 directly binds the LDLR on the cell surface, and is internalized in an LDLR-dependent manner. When the LDLR/PCSK9 complexes reach early endosomes, high percentage of PCSK9-D374Y does not separate from the LDLR, but maintain the binding to the receptor due to 5 to 30-fold greater binding affinity (93, 96). Subsequently, the complex then either goes to lysosomes for degradation or recycles to the cell surface. Images modified from Horton, Cohen, and Hobbs (2009)(72).

77

4.5 A possible role of endosomal calcium concentrations in PCSK9-mediated LDLR

degradation

As discussed in the introduction, PCSK9-mediated LDLR degradation involves the

binding of PCSK9 to the first repeat in the EGF-precursor homology domain of LDLR. This

binding to the EGF-A module is calcium-dependent and enhances in the acidic environment

of early endosomes (91, 92). In the structure of this complex, due to its involvement in the

salt bridge with Arg-194 of PCSK9, Asp-310, which is a calcium-coordinating residue in the

EGF-A repeat, just contributes one of its side-chain oxygen atoms for the calcium

coordination (96). Especially, the calcium coordination observed in the LDLR EGF-A

domain when binding to PCSK9 has similar geometry to the coordination in the EGF-like

domain of C1s, the homologous complement serine proteases (122). However, in the EGF-

like module of coagulation factor IX, the equivalent aspartate residue (Asp-64) coordinates

calcium by both of its side-chain oxygen atoms (123) (Figure 4.3). Moreover, previous work

reported that mutations causing Asp-64 to Asn change in factor IX, which altered the

contribution of residue 64 for the calcium coordination from two side-chain oxygens to one

side-chain oxygen, resulted in approximately 1000-fold lower calcium-affinity of the EGF-

like domain compared to the wild-type, and subsequently, led to a functionally defective

factor IX in hemophilia B patients (112, 124).

Thus, we hypothesize that through forming a salt bridge between its Arg-194 residue

and the side-chain oxygen of LDLR – Asp-310, PCSK9 might induce a conformation change

in the calcium-binding site, which affects titration of calcium of the EGF-A domain in early

endosomes. Especially, it was shown that the measured calcium-affinity in the LDLR EGF-A

domain was closely tuned to endosomal calcium concentrations. The plasma calcium

concentration is ~2 mM, which decreases about 50- to 200-fold within endosomal compartments

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Figure 4.3: Binding of PCSK9-R194 to LDLR-D310 in the calcium-binding site of the LDLR EGF-A domain. The calcium coordination within the PCSK9:EGF-A complex are shown. In the structure of this complex, Arg-194 of PCSK9 forms a salt bridge with EGF-A – Asp-310, a calcium-coordinating residue in the EGF-A repeat. Therefore, Asp-310 just contributes one of its side-chain oxygen atoms for the calcium coordination. The other calcium ligands in the EGF-A module consist of side-chain oxygen from Glu-296; the carbonyl oxygens of Thr-294, Leu-311, and Gly-314; and a water molecule, forming a classic pentagonal bipyramid. Besides, there is a seventh ligand, the carbonyl oxygen of Cys-292 (96). The calcium coordination observed in EGF-A when bound to PCSK9 has the similar geometry to the coordination in the EGF-like domain of C1s (122). However, in the EGF-like module of coagulation factor IX, the equivalent aspartate residue (Asp-64) coordinates calcium by both of its side-chain oxygens (123). Calcium is shown as a green sphere and water molecules are shown as red spheres. The equatorial ligands are indicated by blue dots and the axial ligands are indicated by red dots.

79

depending on the cell-types (125, 126). Therefore, differences in endosomal calcium

concentrations could account for dissimilar responses to PCSK9-mediated LDLR

degradation in hepatic and fibroblast cells. Our working model is that if PCSK9-responsive

cells had higher endosomal calcium concentrations than PCSK9-nonresponsive cells, it

would lead to more LDLRs in which the EGF-A domain is still calcium loaded and PCSK9-

binding competent because PCSK9 binding to the LDLR is calcium-dependent. So, PCSK9

would be able to promote LDLR degradation by maintaining the stable interaction to the

receptor in hepatic cells. While changed calcium titration in the LDLR EGF-A domain due to

PCSK9 binding, which combined with the relatively lower calcium concentrations in early

endosomes of fibroblast cells, could cause loss of calcium from the EGF-A domain and wild-

type PCSK9 dissociation from the LDLR, allowing the receptor to recycle to the cell surface

in these cells. However, the mutant PCSK9-D374Y might dissociate less frequently in

fibroblast cells due to 5 to 30-fold greater affinity in binding to the LDLR versus wild-type

PCSK9.

We expected that changing the interaction between PCSK9 and the LDLR from

calcium-dependent to calcium-independent would allow PCSK9 to maintain the binding to

the receptor, and subsequently, to be effective in degrading LDLRs in fibroblast cells. In the

present study, fibroblast cells expressing the LDLR mutation (LDLR-EGF66) that binds

PCSK9 in a calcium-independent manner (115) did become responsive to wild-type PCSK9

although still in a lesser extent compared with hepatic cells. Surprisingly, addition of the

mutant PCSK9-D374Y to the medium of culture fibroblasts expressing either wild-type

LDLR or LDLR-EGF66 did not result in a significant difference of LDLR levels between

these cells. Instead, they responded equally to PCSK9-D374Y (Figure 3.14). These data

implicated that the LDLR-EGF66 that would presumably help maintain PCSK9 binding to

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the LDLR, even in the lower calcium-concentration environment of fibroblast endosomes,

could partially restore wild-type PCSK9 activity, but not PCSK9-D374Y activity. Therefore,

the maintenance of PCSK9 binding to the LDLR would not be sufficient enough to direct the

receptor to lysosomes for degradation in fibroblast cells. Zhang et al. (2008) supported a

model in which the LDLR/PCSK9 complex might interact with another protein that signals

lysosomal delivery of the complex. In this study, they indicated that although other regions

such as at least three ligand-binding repeats and the β-propeller domain of LDLR as well as

the C-terminal domain of PCSK9 were not required for PCSK9 binding or receptor

internalization, they were required for PCSK9-mediated LDLR degradation (104). Thus, it is

possible that loss of calcium from the EGF-A domain due to changed calcium titration along

with considerably lower calcium concentrations in early endosomes of fibroblast cells could

disrupt the binding interface between the LDLR/PCSK9 complex and the protein required

for lysosomal degradation. Therefore, even when PCSK9-D374Y or the mutant LDLR-

EGF66 still retained the binding, they failed to promote LDLR degradation in fibroblast

cells. In fact, several studies have demonstrated that the calcium-binding site serves a critical

role in maintaining the conformation and functions for diverse proteins containing EGF-like

domain. Alterations in calcium-affinity of the EGF-A module that result in the local

flexibility of this domain can significantly affect the global conformation of the receptor

(127). Additional experiments will be needed not only to further characterize the LDLR-

EGF66 mutation but also to address this possibility.

4.6 Future directions

The current studies not only determined the cell-type specificity of PCSK9 activity but

also suggested the potential importance of endosomal calcium concentrations in PCSK9-

81

mediated LDLR degradation. However, there are still critical aspects related to dissimilar

responses of hepatic cells and other cell-types to PCSK9 that have not been well understood,

highlighting the need for future experiments to further characterize the cell-type dependent

manner of PCSK9 as well as the role of endosomal calcium concentrations in LDLR

degradation mediated by PCSK9.

As stated in the discussion, PCSK9 promoted LDLR degradation in mouse brains

during development and following ischemic stroke whereas it had no effects on LDLR levels

of adult mouse brains (120), suggesting PCSK9 might affect LDLRs differently under

various conditions. Therefore, studies in which PCSK9-responsiveness of more cell lines

from a wide range of conditions are assessed will be needed to address this question.

Our working model for dissimilar responses to PCSK9-mediated LDLR degradation in

different cell types is the endosomal calcium concentration. This hypothesis will be further

verified by examining the effects of altered calcium levels of endosomes on PCSK9-

mediated LDLR degradation in nonresponsive and responsive cells. For example, we

anticipate that using calcium chelators such as BAPTA (1,2-bis(o-aminophenoxy)ethane-

N,N,N’,N’-tetraacetic acid) to specifically decrease free calcium concentrations of

endosomes would make hepatic cells become nonresponsive to PCSK9. In contrast, when

endosomal calcium levels were increased by using bafilomycin, a specific inhibitor of the

vacuolar-type proton ATPase, to block loss of calcium from endosomes (89) or expressing

transient receptor potential mucolipins (TRPMLs), which are calcium-permeable ion

channels within the endolysosomal system, fibroblast cells could turn into PCSK9-

responsive cells.

In addition, we also want to further characterize the effects of LDLR-EGF66 as well as

LDLR-E296Q mutations on LDLR degradation mediated by PCSK9. Indicated in the

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discussion, E296Q mutation would presumably decrease calcium-affinity of the EGF-A

domain more extensively according to the corresponding residue in coagulation factor IX

(112). We expected that hepatic cells expressing this mutation would become PCSK9-

nonresponsive due to the dramatic loss of calcium from the EGF-A module in endosomal

environments that subsequently results to PCSK9 dissociation from the LDLR, even when

these cells have higher levels of calcium in endosomes. However, the characterization of

LDLR-E296Q variation is not a small order because this mutation fails to bind as well as

uptake exogenous PCSK9, highlighting the need for binding studies in order to identify

appropriate conditions by which PCSK9 can be internalized into cells expressing LDLR-

E296Q.

4.7 Conclusion

Following its first discovery in 2003, PCSK9 has become the subject of growing

interest and intense research in cholesterol regulation. Although our understanding of this

proprotein convertase has advanced significantly over the past years, many critical

mechanistic as well as clinical questions still need to be answered. Particularly, the

mechanisms by which PCSK9 promotes LDLR degradation in lysosomes have not been fully

determined. In the first stage, this thesis research aimed to define the cell-type specificity of

PCSK9 activity, and more specifically, to investigate potential factors that result in dissimilar

effects of PCSK9 on LDLRs in different cell-types. Consistent with numerous studies, we

confirmed that PCSK9 caused robust degradation of LDLRs in hepatic cells whereas its

activity was completely ineffective in fibroblast cells. Using these cells as representative

models, we were able to further characterize the fate of PCSK9 after being internalized in

responsive and non-responsive cells. In hepatic cells or responsive cells, our results

supported the model in which PCSK9 is internalized in an LDLR-dependent manner. After

83

the LDLR/PCSK9 complex reaches early endosomes, both of wild-type PCK9 and PCSK9-

D374Y maintain the binding to the LDLR and route the receptor to lysosomes where the

whole complex is degraded, resulting in a significant reduction of LDLR levels in hepatic

cells. Nevertheless, wild-type PCSK9 trafficking is not the same as the mutant in fibroblast

cells. It was shown that wild-type PCSK9 dissociates from the LDLR in early endosomes,

followed by the lysosomal delivery of wild-type PCSK9 while the receptor recycles to the

cell surface. In contrast, higher percentage of the mutant PCSK9-D374Y, which has greater

binding affinity toward the LDLR, was capable of retaining the binding to the receptor in

early endosomes. Subsequently, the LDLR/PCSK9 complex then either goes to lysosomes

for degradation or recycles to the cell surface. Combined together, we conclude that two

factors may diminish PCSK9 activity in fibroblast cells: i) an increased dissociation from the

LDLR in early endosomal compartments as we see in the situation of wild-type PCSK9, and

ii) a decreased ability of bound PCSK9 to direct the LDLR to lysosomes for degradation for

the mutant PCSK9-D374Y. These further insights provide us important cues for future

studies to explore underlying mechanisms by which PCSK9 routes the LDLR to lysosomes

for degradation in hepatic cells, but not in fibroblast cells. For example, the LDLR variation

(EGF66) that binds PCSK9 in a calcium-independent manner could partially restore wild-

type PCSK9 activity, but not PCSK9-D374Y activity, in fibroblast cells, suggesting a

potential role of endosomal calcium concentrations in PCSK9-mediated LDLR degradation.

In fact, by changing the calcium coordination geometry, the binding of PCSK9 could affect

calcium titration of the LDLR EGF-A domain in early endosomes, and different endosomal

calcium concentrations could account for dissimilar responses to PCSK9-mediated LDLR

degradation in hepatic and fibroblast cells. This hypothesis will be addressed in future

studies.

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Curriculum Vitae

Summary

I have been considered as a promising student who possessed valuable abilities and

characteristics to become an outstanding researcher such as energetic, hard working,

intelligent and determined. During my studies, I have set myself up as one of top students

who obtained excellent academic achievements, and granted honorable scholarships. At

work, I have distinguished myself by a high sense of responsibility, creativity, honesty and

strong analytical skills.

Education

§ Master’s in Biochemistry (2011-present)

University of Ottawa – Department of Biochemistry, Microbiology and Immunology

“Characterization of PCSK9-mediated LDLR degradation in hepatic and non-hepatic

cells”

§ Bachelor of Science (Honors) in Biotechnology (2005-2009)

University of Science – Vietnam National University in Ho Chi Minh City

Department of Biotechnology

Distinctions

§ Graduation with Honorable Mention, Magna Cum Laude

University of Science – Vietnam National University in Ho Chi Minh City

(B.Sc. 2005-2009)

Scholarships and Awards

§ FGPS Travel Award – Faculty of Graduate and Postdoctoral Studies, University of

Ottawa (2012)

§ BCH Travel Award – Biochemistry Graduate Program, University of Ottawa

(2012)

§ Ajinomoto’s Scholarship – Ajinomoto Vietnam, Inc. (2008 – 2009)

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§ 3rd prize in “Mendel’s nursery garden” – the annual biology contest at University of

Science, Vietnam National University in Ho Chi Minh City (2008)

§ Encouraging Scholarship – HSBC Bank, Vietnam (2006)

§ Excellence Scholarship – University of Science, Vietnam National University in Ho

Chi Minh City (2005 – 2009)

§ 2nd prize in Ho Chi Minh City biological contest (2005)

§ Encouraging Scholarship – United Airlines (2005)

Conferences and Presentations

§ University of Ottawa Heart Institute Research Day (May 2013)


Poster presentation

§ The Ottawa Heart Research Conference: Emerging Pathways in Cardiovascular

Disease (May 2013)


Attendant

§ University of Ottawa – Department of Biochemistry, Microbiology and

Immunology Seminar Day (March 2013)


Oral presentation

§ “Frontiers in Lipid Biology” – Canadian Lipoprotein Conference (September 2012)

Banff, Alberta, Canada

Poster presentation

§ University of Ottawa – Department of Biochemistry, Microbiology and

Immunology Poster Day (May 2012)


Poster presentation

MY-ANH NGUYEN - University of Ottawa€¦ · MY-ANH NGUYEN . Thesis submitted to the Faculty of...

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